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https://f1000research.com/articles/5-307/v1	09 Mar 16	{ "type": "Research Note", "title": "A metabolite of prostaglandin D2, 11β-prostaglandin F2α (11β-PGF2α), in exhaled breath condensate and serum of asthmatics with airway hyperresponsiveness to distilled water", "authors": [ "Eduard V. Nekrasov", "Juliy M. Perelman", "Denis E. Naumov", "Anna G. Prikhodko", "Elena V. Ushakova", "Victor P. Kolosov", "Juliy M. Perelman", "Denis E. Naumov", "Anna G. Prikhodko", "Elena V. Ushakova", "Victor P. Kolosov" ], "abstract": "This study aims at identifying prostaglandin D2 (PGD2) involvement in osmotic airway hyperresponsiveness of asthmatics. PGD2 primary plasma metabolite, 11β-PGF2α, was analyzed in exhaled breath condensate (EBC) in response to ultrasonically nebulized distilled water (UNDW) and in serum in asthmatics with different airway response to the hypoosmotic stimulus. The total group of asthmatics (n=27) had a lower basal level of 11β-PGF2α (0.38±0.13 pg/ml, mean±SEM) in EBC compared to a group of healthy subjects (0.86±0.31 pg/ml, n=5), which decreased following the UNDW challenge to 0.30±0.09 and 0.53±0.12, respectively. The group of asthmatics with airway hyperresponsiveness to UNDW (≥10% FEV1 drop from baseline, n=14) had a lower concentration of the metabolite (0.28±0.14 pg/ml) as compared to the group without hyperresponsiveness (0.49±0.31 pg/ml, n=10). The 11β-PGF2α concentration decreased in the both groups after the challenge: 0.20±0.04 and 0.23±0.07 pg/ml in the groups with and without hyperresponsiveness to UNDW, respectively . Serum content of 11β-PGF2α was ranging from 0 to 61 pg/ml in asthmatics (n=17) and from 7.3 to 85.4 pg/ml in healthy subjects (n=8). It was lower in the group with airway hyperresponsiveness to UNDW (8.4±1.7 pg/ml, n=9) than in the group without the hyperresponsiveness (21.0±8.8 pg/ml, n=8). The obtained results do not support the involvement of PGD2 in the pathophysiology of asthma with airway hyperresponsiveness to a hypoosmotic stimulus unless other conversions of the prostaglandin occur in the airway under these conditions with formation of metabolites different from 11β-PGF2α.", "keywords": [ "Asthma", "airway osmotic hyperresponsiveness", "exhaled breath condensate", "prostaglandin D2", "11β-prostaglandin F2α" ], "content": "Introduction\n\nProstaglandin D2 (PGD2) is produced by mast cells and macrophages increasing in response to allergen exposure. In asthmatics, PGD2 affects the airways by causing bronchoconstriction, vasodilation, increasing capillary permeability and mucous production1. The role of the prostaglandin in asthmatics with osmotic airway hyperresponsiveness is ill-defined. PGD2 is an unstable compound rapidly metabolized with 11β-PGF2α being its primary plasma metabolite. The aim of this study was estimation of 11β-PGF2α in exhaled breath condensate (EBC) in response to ultrasonically nebulized distilled water (UNDW) and serum in asthmatics with different airway response to the hypoosmotic stimulus.\n\n\nMethods\n\nThe study protocol was approved by the Biomedical Ethics Committee of the Far Eastern Scientific Center of Physiology and Pathology of Respiration (permit #91-1 of 12.01.2015). 39 patients with mild to moderate asthma and 13 healthy subjects participated in the study. The patients were recruited from the Center’s in-patient facilities or invited for follow-up checks. EBC was collected from 27 asthmatics and 5 healthy subjects before and after 3 min provocation with UNDW. Aerosol (the particle size range 0.5–10 μm, average particle size 3 μm) was generated by a Thomex L-2 ultrasonic nebulizer (Poland) operated at 4.5 ml/min. The hypoosmolar challenge test consisted of two consecutive inhalations for 3 min each. The first inhalation was with 30 ml of isotonic solution (0.9% NaCl), and the second with the same volume of distilled water. The temperature of the solution was maintained at 37.3°C. During the inhalation, a participant used a nose-clip and was breathing in a calm manner through a mouthpiece connected via a two-way valve to the container with a solution. Lung function testing was conducted with a spirometer Easy on-PC (ndd Medizintechnik AG, Switzerland) before the provocation test (baseline) and at 1 and 5 min of the recovery period. A drop in forced expiratory volume in 1 sec (ΔFEV1) of 10% or more from baseline after inhalation with UNDW was considered as airway hyperresponsiveness to the hypoosmotic stimulus. Patients who experienced airway responsiveness to isotonic solutions (n=3) were excluded from the test with UNDW, and they were not included in any group except the total group of asthmatics. Patients who experienced any airway responsiveness were given 2 doses of a selective β2-adrenoceptor agonist to prevent bronchoconstriction. EBC was collected during tidal breathing with a condensing device ECoScreen 2 (Erich Jaeger, Germany). Serum was obtained from 17 patients and 8 healthy subjects before the provocation test. The collected samples were stored at -70°C until analysis. The concentration of 11β-PGF2α was measured by 11β-prostaglandin F2α EIA kit (No. 516521, Cayman Chemical Company, USA) in EBC after freeze-drying and in serum after purification by solid phase extraction on C18 cartridges (Strata C18-E, 55 μm, 70A, 500 mg/6 ml, Phenomenex, USA) as recommended by the manufacturer [Cayman’s kit booklet. URL: https://www.caymanchem.com/pdfs/516521.pdf]. The EIA kit utilizes 11β-PGF2α-specific rabbit antiserum, and mouse anti-rabbit monoclonal antibody. Each sample was assayed in duplicate. The optical densities of the samples were used to calculate the concentrations of 11β-PGF2α using an automated Excel spreadsheet [A. Swart 2012–2015. URL: http://www.rheumatologie-neuss.net/index_files/RheumatologieNeuss13.htm].\n\nAnalysis of the data was performed using standard methods of variational statistics. Statistical differences between groups were calculated by Student’s t test or by the nonparametric criteria of Mann-Whitney and Kolmogorov-Smirnov tests in the case of non-Gaussian distribution of variables (Statistica 8.0, StatSoft Inc., Tulsa, OK, USA, 2008).\n\n\nResults\n\nThe level of 11β-PGF2α in EBC was below the announced detection limit (80% B/B0) of the kit (5.5 pg/ml). For the estimation of the 11β-PGF2α level in EBC before and after the provocation test and comparison of the changes in the groups of healthy subjects and asthmatics with and without airway hyperresponsiveness to UNDW, calibration curves were built using the 11β-PGF2α standard in the range of 0–25.6 pg/ml with additional dilutions of the standard down to 0.64 and 0.256 pg/ml and plotting %B/B0 vs. 11β-PGF2α concentration. Obtained values of %B/B0 for the dilutions were different from 100% (Dataset 1, Table 1).\n\nAbbreviations: AHR – airway hyperresponsiveness; EBC – exhaled breath condensate; SEM – standard error of mean; UNDW – ultrasonically nebulized distilled water\n\nAs a result, the calculated content of 11β-PGF2α in EBC was in the range of 0–3.1 pg/ml (Table 1). The total group of asthmatics had a lower basal level of 11β-PGF2α (0.38±0.13 pg/ml, mean±SEM) compared to the group of healthy controls (0.86±0.31 pg/ml), which further decreased following the UNDW challenge to 0.30±0.09 and 0.53±0.12, respectively. The group of asthmatics with airway hyperresponsiveness to UNDW was found to have a lower concentration of the metabolite (0.28±0.14 pg/ml) as compared to the group without the hyperresponsiveness (0.49±0.31 pg/ml). The 11β-PGF2α concentration decreased in both groups after the challenge: 0.20±0.04 and 0.23±0.07 pg/ml in the groups with and without hyperresponsiveness to UNDW, respectively.\n\nThe content of 11β-PGF2α in serum was higher ranging from 0 to 61 pg/ml in the total group of asthmatics and from 7.3 to 85.4 in healthy subjects (Table 1). Once again, it was lower on average in the total group of asthmatics than in the healthy subjects (14.3±4.3 vs. 33.7±12.1 pg/ml), and lower in the group with airway hyperresponsiveness to UNDW (8.4±1.7 pg/ml) than in the group without hyperresponsiveness (21.0±8.8 pg/ml). Due to the high variation of 11β-PGF2α content in the subjects, all differences were statistically insignificant (p>0.05).\n\nSince prostaglandin D2 is considered to be a mast cell- and macrophage-specific eicosanoid, the lack of an increase in the concentration of its major metabolite 11β-PGF2α found in the present study suggests a diminished role of these immune cells in the pathogenesis of the inflammatory reaction in asthma patients with osmotic airway hyperresponsiveness. However, the formation of different metabolites of PGD2, apart from 11β-PGF2α, have been reported2 which may possess different physiological activities.\n\n\nConclusion\n\nThe obtained results do not support the involvement of PGD2 in the pathophysiology of asthma with airway hyperresponsiveness to a hypoosmotic stimulus unless other conversions of the prostaglandin occur in the airway under these conditions with formation of metabolites different from 11β-PGF2α. It would be interesting to investigate the level of other possible metabolites of PGD2.\n\n\nData availability\n\nF1000Research: Dataset 1. Data for calibration curves, 10.5256/f1000research.8084.d1154173\n\nF1000Research: Dataset 2. 11β-PGF2α content (pg/ml) in exhaled breath condensate (EBC) of individual subjects before and after provocation with ultrasonically nebulized distilled water and change in forced expiratory volume in 1 sec (% ΔFEV1 from baseline) after the provocation, 10.5256/f1000research.8084.d1154184\n\nF1000Research: Dataset 3. 11β-PGF2α content (pg/ml) in serum of individual subjects before provocation with ultrasonically nebulized distilled water and change in forced expiratory volume in 1 sec (% ΔFEV1 from baseline) after the provocation (if applicable), 10.5256/f1000research.8084.d1154195\n\n\nConsent\n\nThe study protocol was approved by the Biomedical Ethics Committee of the Far Eastern Scientific Center of Physiology and Pathology of Respiration (permit #91-1 of 12.01.2015), and the participants gave written informed consent.", "appendix": "Author contributions\n\n\n\nAGP – design of the protocol, the recruitment and clinical investigation of the subjects, data collection and analysis, approval of the manuscript. DEN and EVU – method development, data acquisition, approval of the manuscript. EVN – sample processing (lyophilisation, purification by SPE), data analysis, drafting of the manuscript. JMP – leader of the project, conception and design of the study, critical revision of the manuscript, final approval of the version. VPK – conception of the study, final approval of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe research was supported by Russian Scientific Foundation (grant No.14-25-00019 was awarded to the Far Eastern Scientific Center of Physiology and Pathology of Respiration, the leader of the project is Dr. J.M. Perelman).\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgement\n\nThe authors are thankful to Galina A. Makarova (FESC PPR) for technical assistance.\n\n\nReferences\n\nOguma T, Asano K, Ishizaka A: Role of prostaglandin D2 and its receptors in the pathophysiology of asthma. Allergol Int. 2008; 57(4): 307–312. PubMed Abstract \| Publisher Full Text\n\nSchuligoi R, Schmidt R, Geisslinger G, et al.: PGD2 metabolism in plasma: kinetics and relationship with bioactivity on DP1 and CRTH2 receptors. Biochem Pharmacol. 2007; 74(1): 107–117. PubMed Abstract \| Publisher Full Text\n\nNekrasov EV, Perelman JM, Naumov DE, et al.: Dataset 1 in: A metabolite of prostaglandin D2, 11β-prostaglandin F2α (11β-PGF2α), in exhaled breath condensate and serum of asthmatics with airway hyperresponsiveness to distilled water. F1000Research. 2016. Data Source\n\nNekrasov EV, Perelman JM, Naumov DE, et al.: Dataset 2 in: A metabolite of prostaglandin D2, 11β-prostaglandin F2α (11β-PGF2α), in exhaled breath condensate and serum of asthmatics with airway hyperresponsiveness to distilled water. F1000Research. 2016. Data Source\n\nNekrasov EV, Perelman JM, Naumov DE, et al.: Dataset 3 in: A metabolite of prostaglandin D2, 11β-prostaglandin F2α (11β-PGF2α), in exhaled breath condensate and serum of asthmatics with airway hyperresponsiveness to distilled water. F1000Research. 2016. Data Source" }	[ { "id": "17321", "date": "01 Nov 2016", "name": "Andras Bikov", "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 read the article with great interest. Lipid metabolites play a role in the pathophysiology of asthma and their changes during different stimuli are of interest.\nThe critical point of the article is that the EBC 11β-PGF2α levels were below the lower detection limit of the ELISA kit used in the study. Thus, the authors can simply not conclude on the results as they might have experienced changes and differences on variations of zero (distilled water)... To resolve this crucial error, I recommend concentrating the sample (i.e. vacuum evaporation), or adding a known concentration of standard to each sample to overcome the detection limit.\n\nThe article is too short. Methodology of EBC collection is extremely variable, and to compare results with external findings, EBC methodology needs to be decribed in more detail.", "responses": [] }, { "id": "20065", "date": "09 Feb 2017", "name": "Giuseppe Santini", "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 critical point of this article is the detection limit of the ELISA KIT used. The concentration of metabolite measured in EBC was too much low. I suggest to concentrate the sample by evaporation under vacuum, or use an another KIT with a higher sensitivity.\n\nThe number of healthy subjects from which it was collected EBC is too low. I suggest of collect others EBC samples from healthy subjects.", "responses": [] } ]	1	https://f1000research.com/articles/5-307
https://f1000research.com/articles/5-99/v1	22 Jan 16	{ "type": "Research Note", "title": "The importance of selecting the appropriate reference genes for quantitative real time PCR as illustrated using colon cancer cells and tissue", "authors": [ "Catríona M. Dowling", "Dara Walsh", "John C. Coffey", "Patrick A. Kiely", "Dara Walsh", "John C. Coffey" ], "abstract": "Quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR) remains the most sensitive technique for nucleic acid quantification. Its popularity is reflected in the remarkable number of publications reporting RT-qPCR data. Careful normalisation within RT-qPCR studies is imperative to ensure accurate quantification of mRNA levels. This is commonly achieved through the use of reference genes as an internal control to normalise the mRNA levels between different samples. The selection of appropriate reference genes can be a challenge as transcript levels vary with physiology, pathology and development, making the information within the transcriptome flexible and variable. In this study, we examined the variation in expression of a panel of nine candidate reference genes in HCT116 and HT29 2-dimensional and 3-dimensional cultures, as well as in normal and cancerous colon tissue. Using normfinder we identified the top three most stable genes for all conditions. Further to this we compared the change in expression of a selection of PKC coding genes when the data was normalised to one reference gene and three reference genes. Here we demonstrated that there is a variation in the fold changes obtained dependent on the number of reference genes used. As well as this, we highlight important considerations namely; assay efficiency tests, inhibition tests and RNA assessment which should also be implemented into all RT-qPCR studies. All this data combined demonstrates the need for careful experimental design in RT-qPCR studies to help eliminate false interpretation and reporting of results.", "keywords": [ "Quantitative real-time PCR", "Normalisation", "Reference Genes", "NormFinder", "Colon Cancer" ], "content": "Introduction\n\nGene expression analysis is a critical and important tool in molecular diagnostics and medicine1–4. Quantification of RNA transcripts is carried out using one of four common methods; reverse transcription polymerase chain reaction (RT-PCR)5, RNase protection assays6, northern blotting and in situ hybridisation7, and less commonly now using cDNA arrays8. At present, the most popular and widely used method for gene expression is fluorescence based quantitative real time PCR (RT-qPCR)9. It is the most sensitive and flexible of the quantitative methods with a capacity to detect and measure minute amounts of nucleic acids10,11. There are two types of quantitative methods that can be applied within RT-qPCR; absolute quantification and relative quantification. Absolute quantification relates the PCR signal to a standard curve to determine the input copy number of the gene of interest. In contrast, relative quantification evaluates the change in expression of a target gene relative to a reference group, for example an untreated control12.\n\nWhen employing RT-qPCR to compare mRNA levels between two different test conditions, it is imperative that reference genes are utilised carefully9,10. Normalisation of the data with these reference genes is essential for correcting results of different amounts of input RNA, uneven loading, reverse-transcription yield, efficiency of amplification and variation within experimental conditions9,13. The mRNA of reference genes should be stably expressed and their expression should not be affected by experimental condition or by any human disease14. Numerous studies have demonstrated that common reference genes, such as β-Actin and GAPDH, which are largely accepted as being stably expressed within cells, can in fact show large variations in expression15–18. Despite the awareness that validation of the stability of reference genes is an essential component for accurate RT-qPCR analysis, this consideration is still largely disregarded19–21.\n\nFurther to this, it is reported that over 90% of gene expression analysis published in high impact journals used only one reference gene22–24. It has since been widely documented that normalisation of data with a single reference gene can lead to inaccurate interruption of results10,25,26. Taken together, this highlights the importance of selecting the optimal number and type of reference genes for any RT-qPCR study. Other essential considerations such as; analysis of assay efficiency, testing for inhibition with biological samples and reporting the quality and integrity of input RNA are all highlighted in the ‘MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments’10.\n\nIn this study, we sought to highlight the importance of carefully-designed RT-qPCR studies in order to avoid the reporting of inaccurate and misleading information. We test a panel of nine candidate reference genes and report their stability between 2-dimensional and 3-dimensional HCT116 and HT29 colon cancer cell lines, as well as between normal and cancerous tissue from colon cancer patients. We also demonstrate useful tests that should be implemented within RT-qPCR studies to ensure that studies comply with the MIQE guidelines.\n\n\nMethods\n\nHCT116 (ATCC® CCL-247™) and HT29 (ATCC® HTB-38™) cell lines were obtained from ATCC. These cell lines were cultured in complete Dulbecco's modified essential medium (DMEM) supplemented with 10% of foetal bovine serum, 1% of penicillin/streptomycin and 1% of L-glutamine. All cells were incubated at 37°C in a humidified 95% air/5% CO2 environment. Cellular suspensions were obtained by adding 0.5% trypsin to the cultures and incubating at 37°C at 5% CO2.\n\nIndividual wells of a 6-well plate were coated with MatrigelTM (BD Biosciences) and placed in an incubator at 37°C for 30 min. Cell lines were trypsinized and counted. 50,000 cells/ml were resuspended in DMEM supplemented with 2% MatrigelTM. Cells were placed in MatrigelTM coated wells for 30 min at 37°C, after which DMEM supplemented with 2% MatrigelTM was added to the cultures. Cells were maintained in culture for 6 days in an incubator at 37°C, 5% CO2 with fresh medium added every 2 days. On day 6, cultures were harvested using EDTA/PBS and either fixed with paraformaldehyde (PFA) for confocal analysis (Zeiss LSM 710) or used for RNA extraction.\n\nFollowing ethical approval from the University Hospital Limerick’s Ethics Committee (ethical approval number 73/11), tissue samples measuring approximately 0.5cm in diameter were collected from patients undergoing surgery in University Hospital Limerick. Normal tissue from the patients was also collected approximately 10 cm away from the cancer tissue. Specimens were immediately placed in Allprotect tissue reagent (Qiagen) and stored at -80°C.\n\n2-dimensional and 3-dimensional cell cultures were trypsinised as described above and frozen tissue was immersed in liquid nitrogen and ground into powder. Lysis buffer was added to the cells and tissue and the samples transferred to tubes using a 21-gauge needle. Total RNA was extracted as per Qiagen RNeasy Mini Kit instructions. RNA was quantified using a Nanodrop Spectrophotometer (Thermo Scientific) and stored at -80 degrees. RNA purity was evaluated by the ratio of absorbance at 260/280 nm and RNA quality was evaluated through visualization of the 28S:18S ribosomal RNA ratio on a 1% agarose gel. Total RNA (1 μg) was synthesised into cDNA using Vilo cDNA synthesis kit (Invitrogen) and stored at -20 degrees.\n\nReal-time PCR was conducted using the ABI 7900 HT instrument (Applied Biosystems) following supplier instructions. Taqman® Gene Expression Assay Kits (Applied Biosystems) were used to analyse the gene expression of PKC coding genes. Data was normalised to either one reference gene or three reference genes (see below).\n\nThe efficiency of each assay was determined by means of a calibration curve with the logarithm of the initial template concentration plotted on the x axis and the Cq plotted on the y axis. The slope of the graph was obtained and the PCR efficiency was calculated using the equation: 10-1/slope-1.\n\nReal-time PCR was conducted on corn DNA using a corn gene assay with a known Cq value of 24–26. Samples of cDNA from 2D and 3D HCT116 and HT29 cultures and from patient tissue was added to the reaction to test for an inhibitory components that may be present in these biological samples.\n\nAll nine reference genes (Table 1) were purchased as pre-designed Taqman® Gene Expression Assays. The Cq value of each reference gene was determined for all biological samples. Normfinder was used to determine the most stable reference genes between 2D and 3D cell cultures as well as between normal and cancer tissue. Differences in gene expression levels of the PKC coding genes was determined using Pair Wise Fixed Reallocation Randomisation Test© as per REST© software. Within the software data was normalised to either the top reference gene or the top three reference genes.\n\nThe accession numbers for each gene are taken from the National Center for Biotechnology Information.\n\n\nResults\n\nIn this study, we wanted to compare and validate the stability of reference genes used in quantitative real time PCR (RT-qPCR). To do this, HCT116 cells were grown in 2-dimensional and 3-dimensional cultures (Figure 1A). Following this, RNA was extracted from the cultures and cDNA was synthesised. Quantitative real time PCR was utilised to measure the variability in RNA transcript levels of 9 reference genes (RG) (Table 1) in the 2-dimensional and 3-dimensional cultures. The expression levels of the candidate reference genes were determined using the raw Cq values and NormFinder was then utilised to verify the stability of the genes. Normfinder ranks the RGs according to their stability values under the tested conditions. The top three stable genes when comparing 2-Dimensional and 3-dimensional HCT116 cultures were B2M, PMM1 and RPLPO, with B2M and PMM1 showing identical stability levels (Figure 1B, Dataset 1). Next, we wanted to elucidate the benefit of normalising data to more than one RG. To do this, we compared the expression of seven PKC coding genes in 3-Dimensional HCT116 cultures compared to 2-dimensional HCT116 cultures. The data was normalised to either one RG, B2M, or normalised to three RGs, B2M, PMM1 and RPLPO (Figure 1C, Dataset 2). Results indicate that using one RG gives fold changes that are greater than the fold changes obtained using three RGs.\n\nThe stability of the nine candidate reference genes between 2D and 3D HCT116 cultures was analysed using NormFinder. (A) Immunofluorescence images of HCT116 cells in 2D (100X) (left panel) and 3D (right panel) cell cultures (63X). (B) Table displaying the stability levels of the nine candidate reference genes between the 2D and 3D cultures. (C) Graph representing the fold change of PKC coding genes in 3D cultures compared to 2D cultures when using one reference gene (B2M) versus three reference genes (B2M, PMM1 and RPLPO).\n\nNext, we compared the stability of the same 9 candidate reference genes in HT29 cultures. The cells were grown in 2-dimensional and 3-dimensional cultures (Figure 2A) before using RT-qPCR to determine the stability of the RGs between the two conditions. Normfinder revealed the most stable RGs were PMM1, HRPTI, PP1A and TBP (Figure 2B, Dataset 3) with PMM1 and HRPTI having a value of 0.001 and PP1A and TBP having a value of 0.002. Again, we examined the expression of the PKC coding genes in the cultures and normalised the data to one RG, PMM1, or three RGs, PMM1, HRPTI and PP1A (Figure 2C, Dataset 4). Our results indicate that there is variation in the fold changes obtained when using one RG versus three RGs. In some instances, genes that are found to be down-regulated when normalising with one RG are in fact up-regulated when normalising with three RGs.\n\nThe stability of the nine candidate reference genes between 2D and 3D HT29 cultures was analysed using NormFinder. (A) Immunofluorescence images of HT29 cells in 2D (100X) (left panel) and 3D (right panel) cell cultures (63X). (B) Table displaying the stability levels of the nine candidate reference genes between the 2D and 3D cultures. (C) Graph representing the fold change of PKC coding genes in 3D cultures compared to 2D cultures when using one reference gene (PMM1) versus three reference genes (PMM1, HRPT1 and PP1A).\n\nFollowing this, we wanted to examine the stability of the nine candidate RGs in normal and colon cancer tissue. We used fresh tissue samples that were excised from both the cancer tissue and normal distant tissue of individual patients (Figure 3A). As above, the expression levels of the nine candidate RGs were determined and Normfinder was used to establish the stability of the genes. PGK1, GUSB and PP1A were ranked as the most stable genes between normal and cancerous tissue (Figure 3B, Dataset 5). Next, we examined the change in PKC coding genes in colon cancer tissue when the data was normalised to one RG, PGK1, and normalised to three RGs, PGK1, GUSB and PP1A (Figure 3C, Dataset 6). The results demonstrate that using one RG can present fold changes that are up to 2-fold greater than when using three RGs.\n\nThe stability of the nine candidate reference genes between normal and cancer tissue was analysed using NormFinder. (A) Surgical image of specimen resected from a colon cancer patient. (B) Table displaying the stability levels of the nine candidate reference genes between the normal and cancer tissue. (C) Graph representing the fold change of PKC coding genes in cancer tissue compared to normal tissue (n=21) when using one reference gene (PGK1) versus three reference genes (PGK1, GUSB and PP1A).\n\nTaken together, the results indicate that variations in fold changes can occur depending on the RG used to normalise data; making the selection of the correct RGs an imperative part of RT-qPCR studies. Further to this, the testing and reporting of assay efficiency is also essential to prevent the reporting of misinformation. Taking this into consideration, we examined the efficiency of all the RG assays and PKC coding genes assays (Figure 4A, Dataset 7). This information was inputted into the REST© software when establishing changes in gene expression between tested conditions. Another important consideration when designing RT-qPCR studies is the testing of your cDNA for any contaminants which could lead to the inhibition of the RT-qPCR reaction. For this reason, we added our samples to a standard RT-qPCR reaction using corn DNA and a gene that is known to have a Cq value of 24–26. If there were contaminants present in our cDNA samples this would inhibit the reaction resulting in a reduction in the Cq values10. However, we found no change in the Cq values for the reactions with the cDNA added, indicating the samples do not have any contaminates that will affect the amplification of our genes (Figure 4B, Dataset 8). It is also essential to report the quality assessment of the RNA templates, such as the RNA quantity, quality and integrity. We evaluated the RNA purity by the ratio of absorbance at 260/280 nm and RNA quality was assessed through visualization of the 28S:18S ribosomal RNA ratio on a 1% agarose gel (Figure 4C,D).\n\n(A) Representative graph of assay efficiency check. (B) Graph representing the inhibition test for all biological samples. (C) Representative graph from Nanodrop Spectrophotometer displaying the quantity and purity of the RNA. (D) Representative image of agarose gel displaying the 28S:18S ribosomal RNA ratio for RNA samples.\n\n\nDiscussion\n\nThe first publications using fluorescence-based quantitative real time PCR (RT-qPCR)27–30 emerged almost a decade ago and since this time it has become the leading technique for gene expression analysis31,32. While RT-qPCR remains the most sensitive method for the detection of RNA transcripts33 there are also many challenges associated with the technique34,35. One of the major difficulties is the selection of appropriate reference genes for the normalisation of data. Hence the purpose of this study was to evaluate the stability in expression of nine candidate reference genes in two colon cancer cell lines as well as in normal and cancerous tissue from colon cancer patients. To help find the most suitable reference genes we selected genes which display a variation of functions within cells (Table 1).\n\nFirstly, we examined the stability of the nine candidate reference genes between 2-dimensional and 3-dimensional HCT116 and HT29 cultures (Figure 1A, Figure 2A). The use of 3-dimensional cell cultures as cancer models is becoming increasingly popular36–38; making the availability of appropriate reference genes important to help reduce the reporting of misinformation. When we examined the variation in expression between 2-dimensional and 3-dimensional HCT116 cells we found B2M, RPLPO and PMM1 to be the most stable genes between these two conditions (Figure 1B). Many publications have highlighted the problems associated with normalisation of data using only one reference gene22,23, for this reason we wanted to investigate differences in fold changes associated with normalising data to one reference gene compared to three reference genes. To do this, we investigated the change in expression in a selection of protein kinase c (PKC) coding genes between 2-dimensional and 3-dimensional HCT116 cultures. We examined PKCs as they are a group of proteins that are extensively studied for their role in oncogenic signalling39. Interestingly, when normalising the data to the reference gene B2M alone we found the change in expression of PKC coding genes was greater compared to normalisation with the reference genes, B2M, RPLPO and PMM1 together (Figure 1C). This finding highlights the need for normalisation with more than one reference gene to help eliminate the misinterpretation of fold changes in target genes.\n\nNext we wanted to establish the stability of these reference genes in 2-dimensional and 3-dimensional HT29 cultures (Figure 2A). Normfinder ranked PMM1, HRPTI, PP1A and TBP as the most stable genes between these cultures (Figure 2B). It is important to note that despite the fact the treatments here were the same; there was a difference in the selected reference genes for HCT116 and HT29 cultures. This again emphasises the need to conduct stability tests on a panel of reference genes prior to all RT-qPCR studies to ensure data is normalised correctly. Again, we examined the difference in fold changes of PKC coding genes when normalising with varying numbers of reference genes. Importantly, we found that some target genes showing a down regulation when normalised with PMM1 showed no change when normalised to PMM1, HRPT1 and PP1A (Figure 2C).\n\nRT-qPCR is the most common method used for the quantification of individual genetic differences in normal versus cancerous tissue9,34. Recent publications demonstrated that 97% of RT-qPCR studies contained on colorectal cancer contained information that was unreliable21. Thus, when examining difference in mRNA levels between normal and diseased tissue it is imperative the correct reference genes are used to normalise the data to prevent the presence of misleading information in the literature. Using normal and cancer tissue from CRC patients (Figure 3A) we examined the stability of the nine candidate reference genes, finding PGK1, GUSB and PP1A to be the most stably expressed (Figure 3B). As before, we compared the expression of PKC coding genes in normal and cancer tissue with the data normalised to either PGK1 alone or PGK1, GUSB and PP1A together. Strikingly we found that using only one reference gene results in a fold change that is up to 2 fold greater than when using three reference genes. This is a very important observation as it clearly displays that the misuse of reference genes could lead to the incorrect reporting of a dysregulated genes in cancerous tissue.\n\nAlthough the selection of the correct reference genes is a key challenge when conducting RT-qPCR studies there are other aspects of experimental design that also need to be considered10. In this study, we highlighted appropriate tests to comply with necessary measures for RT-qPCR studies (Table 2). When utilising relative quantification it is essential that the gene assay of the reference gene and the target gene are amplified with comparable efficiencies34. For this reason, we examined the efficiency of all gene assays using a calibration curve (Figure 4A) and we used this value when evaluating the fold change between conditions. Another important consideration in experimental design is establishing the presence or absence of biological contaminants in samples which may inhibit the RT-qPCR reaction40. We designed an inhibition assay test and displayed that there was no inhibitors present in any of the samples (Figure 4B). Finally, the documenting of the quality assessment of RNA templates is critical within RT-qPCR studies as it has been observed that there is a difference in gene expression stability between intact and degraded RNA samples from the same tissue and higher gene-specific variation in degraded samples34,41. In this study, we documented the RNA purity by the ratio of absorbance at 260/280 nm and RNA quality through visualization of the 28S:18S ribosomal RNA ratio on a 1% agarose gel (Figure 4C,D).\n\nOur data clearly demonstrates that the variability in the expression of reference genes can lead to false interpretation of results; making the selection of the correct genes essential when normalizing RNA concentrations in RT-qPCR analyses. Further to this we have demonstrated appropriate tests to create studies which comply with the MIQE guidelines. The implementation of these guidelines10,42 should be employed by all reviewers when accepting gene expression studies for publication as it will help eliminate the reporting of inaccurate and misleading information.\n\n\nData availability\n\nF1000Research: Dataset 1. Cq Values for reference genes in HCT116 cell lines, 10.5256/f1000research.7656.d11180343\n\nF1000Research: Dataset 2. Cq Values for PKC coding genes and reference genes in HCT116 cell lines, 10.5256/f1000research.7656.d11180444\n\nF1000Research: Dataset 3. Cq Values for reference genes in HT29 cell lines, 10.5256/f1000research.7656.d11180545\n\nF1000Research: Dataset 4. Cq Values for PKC coding genes and reference genes in HT29 cell lines, 10.5256/f1000research.7656.d11180746\n\nF1000Research: Dataset 5. Cq Values for reference genes in normal and colon cancer tissue, 10.5256/f1000research.7656.d11180847\n\nF1000Research: Dataset 6. Cq Values for PKC coding genes and reference genes in normal and colon cancer tissue, 10.5256/f1000research.7656.d11180948\n\nF1000Research: Dataset 7. Cq values for sample assay efficiency test, 10.5256/f1000research.7656.d11181049\n\nF1000Research: Dataset 8. Cq values for inhibition assay test, 10.5256/f1000research.7656.d11181150\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patients.\n\n(Ethical approval number 73/11, University Hospital Limerick, Limerick, Ireland).", "appendix": "Author contributions\n\n\n\nCMD conducted experimental work and writing of the manuscript. DW provided surgical images of colon tissue. JCC provided normal and colon cancer tissue. PAK reviewed experimental design and writing of manuscript.\n\n\nCompeting interests\n\n\n\nThe authors declare there is no conflict of interest.\n\n\nGrant information\n\nThis work was supported by grants received from the Irish Cancer Society Grant CRS12DOW (to CD), the Mid-Western Cancer Foundation and funding from Science Foundation Ireland grant 13/CDA/2228 (to PK).\n\n\nAcknowledgments\n\nWe are grateful to our colleagues in the Laboratory of Cellular and Molecular Biology for helpful discussions and critical review.\n\n\nReferences\n\nKubista M, Andrade JM, Bengtsson M, et al.: The real-time polymerase chain reaction. Mol Aspects Med. 2006; 27(2–3): 95–125. PubMed Abstract \| Publisher Full Text\n\nRamachandran C, Melnick SJ: Multidrug resistance in human tumors—molecular diagnosis and clinical significance. Mol Diagn. 1999; 4(2): 81–94. 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PubMed Abstract \| Publisher Full Text\n\nHeid CA, Stevens J, Livak KJ, et al.: Real time quantitative PCR. Genome Res. 1996; 6(10): 986–994. PubMed Abstract \| Publisher Full Text\n\nHiguchi R, Fockler C, Dollinger G, et al.: Kinetic PCR analysis: real-time monitoring of DNA ampliﬁcation reactions. Biotechnology (N Y). 1993; 11(9): 1026–1030. PubMed Abstract \| Publisher Full Text\n\nVanGuilder HD, Vrana KE, Freeman WM: Twenty-five years of quantitative PCR for gene expression analysis. Biotechniques. 2008; 44(5): 619–26. PubMed Abstract \| Publisher Full Text\n\nBustin SA: Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol. 2000; 25(2): 169–193. PubMed Abstract \| Publisher Full Text\n\nNolan T, Hands RE, Bustin SA: Quantification of mRNA using real-time RT-PCR. Nat Protoc. 2006; 1(3): 1559–1582. PubMed Abstract \| Publisher Full Text\n\nBustin SA: Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol. 2002; 29(1): 23–39. PubMed Abstract \| Publisher Full Text\n\nBustin SA, Nolan T: Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction. J Biomol Tech. 2004; 15(3): 155–66. PubMed Abstract \| Free Full Text\n\nDebnath J, Brugge JS: Modelling glandular epithelial cancers in three-dimensional cultures. Nat Rev Cancer. 2005; 5(9): 675–688. PubMed Abstract \| Publisher Full Text\n\nMullins SR, Sameni M, Blum G, et al.: Three-dimensional cultures modeling premalignant progression of human breast epithelial cells: role of cysteine cathepsins. Biol Chem. 2012; 393(12): 1405–1416. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDeevi RK, Cox OT, O'Connor R: Essential function for PDLIM2 in cell polarization in three-dimensional cultures by feedback regulation of the β1-integrin-RhoA signaling axis. Neoplasia. 2014; 16(5): 422–431. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDowling CM, Kiely PA: Targeting Protein Kinase C Downstream of Growth Factor and Adhesion Signalling. Cancers (Basel). 2015; 7(3): 1271–1291. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTaylor S, Wakem M, Dijkman G, et al.: A practical approach to RT-qPCR-Publishing data that conform to the MIQE guidelines. Methods. 2010; 50(4): S1–S5. PubMed Abstract \| Publisher Full Text\n\nFleige S, Pfaffl MW: RNA integrity and the effect on the real-time qRT-PCR performance. Mol Aspects Med. 2006; 27(2–3): 126–139. PubMed Abstract \| Publisher Full Text\n\nBustin SA, Beaulieu JF, Huggett J, et al.: MIQE précis: Practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments. BMC Mol Biol. 2010; 11(1): 74. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDowling CM, Walsh D, Coffey JC, et al.: Dataset 1 in: The importance of selecting the appropriate reference genes for quantitative real time PCR as illustrated using colon cancer cells and tissue. F1000Research. 2016. Data Source\n\nDowling CM, Walsh D, Coffey JC, et al.: Dataset 2 in: The importance of selecting the appropriate reference genes for quantitative real time PCR as illustrated using colon cancer cells and tissue. F1000Research. 2016. Data Source\n\nDowling CM, Walsh D, Coffey JC, et al.: Dataset 3 in: The importance of selecting the appropriate reference genes for quantitative real time PCR as illustrated using colon cancer cells and tissue. F1000Research. 2016. Data Source\n\nDowling CM, Walsh D, Coffey JC, et al.: Dataset 4 in: The importance of selecting the appropriate reference genes for quantitative real time PCR as illustrated using colon cancer cells and tissue. F1000Research. 2016. Data Source\n\nDowling CM, Walsh D, Coffey JC, et al.: Dataset 5 in: The importance of selecting the appropriate reference genes for quantitative real time PCR as illustrated using colon cancer cells and tissue. F1000Research. 2016. Data Source\n\nDowling CM, Walsh D, Coffey JC, et al.: Dataset 6 in: The importance of selecting the appropriate reference genes for quantitative real time PCR as illustrated using colon cancer cells and tissue. F1000Research. 2016. Data Source\n\nDowling CM, Walsh D, Coffey JC, et al.: Dataset 7 in: The importance of selecting the appropriate reference genes for quantitative real time PCR as illustrated using colon cancer cells and tissue. F1000Research. 2016. Data Source\n\nDowling CM, Walsh D, Coffey JC, et al.: Dataset 8 in: The importance of selecting the appropriate reference genes for quantitative real time PCR as illustrated using colon cancer cells and tissue. F1000Research. 2016. Data Source" }	[ { "id": "12053", "date": "02 Feb 2016", "name": "Gary Loughran", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDowling et al. reinforce the necessity of using more than one reference gene (RG) for qRT-PCR. They show that not only should more than one RG be used for normalisation but that a panel of RGs should be tested at an early stage to identify the most stable group. They demonstrate clearly how perilous choosing a single inappropriate RG can produce anomalous data.While this study was well designed and well performed there are some omissions that would enhance the report by facilitating repetition by others. It would be nice to see a table listing the primer sequences used, expected amplicon size and whether any particular primer pairs are intron spanning. This would be especially useful for the RGs.One other minor point. Presumably testing the integrity of RNA on a 1% agarose gel was under denaturing conditions (e.g. formaldehyde)?", "responses": [ { "c_id": "1800", "date": "05 Feb 2016", "name": "Catríona Dowling", "role": "Author Response", "response": "We thank the reviewer for the suggestion.The sequences of the primers and probes we used in our assays are pre-designed and are the proprietary of Life Technologies who are unable to release this information to us. However, as stated within the MIQE guidelines, it is acceptable to use the unique assay ID for each TaqMan assay in place of the primer and probe sequences. We are grateful to the reviewer for the suggestion to include such information and we will include a table which displays assay ID, exon boundary and amplicon size for all reference genes.The agarose gels that we ran were non-denaturing gels. We will add this in to the text and to avoid any confusion." } ] }, { "id": "12476", "date": "24 Feb 2016", "name": "Verónica Ayllón Cases", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 Dowling et al. demonstrates the importance of choosing the right reference genes (RG) when performing RT-qPCR experiments. They have compared the effect of using a single RG versus three RGs on the gene expression values of the interrogated genes on a given experiment. They present data that confirms that using a single RG usually gives greater changes in gene expression than when using a panel of three RGs. As a consequence, many published studies that present RT-qPCR results based on a single RG may have over-estimated gene expression changes and generated misleading results.As a conclusion of their work, they present a very useful checklist for any researchers that want to perform gene expression analysis using RT-qPCR, which includes all the steps to follow when designing RT-qPCR experiments.When reviewing this work, there are several minor points that have raised my concern, although they don’t affect the main conclusions of the work. These minor points are:It is not clear to me whether the three Cq values given on the data sets correspond to three independent experiments (biological replicates) or they are three values obtained from the same sample (experimental replicates). In Figure 2C the authors have presented their data in a way that I consider it magnifies their results and it can be slightly misleading. The authors have plotted the fold changes in PKC genes using what is known as “Fold Regulation”, in which the values of Fold Change below 1 are plotted as negative values. When the data is plotted in this way, the area of the graph between +1 and -1 it simply doesn’t exist; the values will always “jump” from >+1 to <-1.If the data presented in Figure 2C was plotted without making this conversion to Fold Regulation we will be able to appreciate more clearly that the expression of several PKC genes does not change much – the values will probably oscillate between 1.2 and 0.8.My recommendation for the authors is to change the way the present their data in this case where the Fold Regulation data oscillates between positive and negatives values, but they are all very close to 1 (that is, there is only a limited variation in expression relative to the control sample of 2D cultures). I suggest two alternatives:- Remove the gap between +1 and -1 in your Y axis.- Present your data as Fold Change, without converting it to Fold Regulation.As a final comment, also please put your gene names in a way that they don’t overlap with the bars, as it is very difficult to read them. This also applies to Figure 3C. Regarding the assessment of RNA purity and integrity, the authors have used spectrophotometry and running an agarose gel, respectively. This is correct, but if we want to compare RNA integrity across samples it would be better to perform this type of analysis using a Bioanalyzer (Agilent Technologies). With this assay we will be able to obtain a more quantitative measurement of RNA integrity in the form of the RIN value. My suggestion to the authors is, if possible, to complement the data they already have with a Bioanalyzer analysis and the corresponding RIN data. In this way they will confirm that the simpler strategy that they propose is a valid one.", "responses": [ { "c_id": "1842", "date": "09 Mar 2016", "name": "Catríona Dowling", "role": "Author Response", "response": "We thank the reviewer for the suggestions. We have adjusted the manuscript accordingly and we agree that it is better presented now.The values given on the datasets corresponds to three independent experiments i.e three biological replicates. We have now written a note within each dataset to clear any ambiguity.We are grateful to the reviewer for making the observation on Figure 2C. We have now updated the figure and removed the area between 1 and -1. We have also changed the layout in our graphs to ensure the gene names don’t overlap with the bars.We agree with the reviewer, using a Bioanalyzer is the preferable method for looking at RNA integrity and purity. Unfortunately, it is not possible for us to complement our data with a Bioanalyzer analysis as we don’t have access to this piece of equipment. We used non-denaturing agarose gels and a nanospectrometer for our work. There are many Universities that do not have access to a Bioanalyzer and for this reason we wanted to offer this alternative approach to encourage all researchers to carry out integrity and purity tests in the absence of such equipment." }, { "c_id": "1848", "date": "09 Mar 2016", "name": "Catríona Dowling", "role": "Author Response", "response": "We thank the reviewer for the suggestions. We have adjusted the manuscript accordingly and we agree that it is better presented now.The values given on the datasets corresponds to three independent experiments i.e three biological replicates. We have now written a note within each dataset to clear any ambiguity.We are grateful to the reviewer for making the observation on Figure 2C. We have now updated the figure and removed the area between 1 and -1. We have also changed the layout in our graphs to ensure the gene names don’t overlap with the bars.We agree with the reviewer, using a Bioanalyzer is the preferable method for looking at RNA integrity and purity. Unfortunately, it is not possible for us to complement our data with a Bioanalyzer analysis as we don’t have access to this piece of equipment.We used non-denaturing agarose gels and a nanospectrometer for our work. There are many Universities that do not have access to a Bioanalyzer and for this reason we wanted to offer this alternative approach to encourage all researchers to carry out integrity and purity tests in the absence of such equipment." } ] } ]	1	https://f1000research.com/articles/5-99
https://f1000research.com/articles/5-306/v1	09 Mar 16	{ "type": "Review", "title": "Airway hyperresponsiveness; smooth muscle as the principal actor", "authors": [ "Anne-Marie Lauzon", "James G. Martin", "Anne-Marie Lauzon" ], "abstract": "Airway hyperresponsiveness (AHR) is a defining characteristic of asthma that refers to the capacity of the airways to undergo exaggerated narrowing in response to stimuli that do not result in comparable degrees of airway narrowing in healthy subjects. Airway smooth muscle (ASM) contraction mediates airway narrowing, but it remains uncertain as to whether the smooth muscle is intrinsically altered in asthmatic subjects or is responding abnormally as a result of the milieu in which it sits. ASM in the trachea or major bronchi does not differ in its contractile characteristics in asthmatics, but the more pertinent peripheral airways await complete exploration. The mass of ASM is increased in many but not all asthmatics and therefore cannot be a unifying hypothesis for AHR, although when increased in mass it may contribute to AHR. The inability of a deep breath to reverse or prevent bronchial narrowing in asthma may reflect an intrinsic difference in the mechanisms that lead to softening of contracted ASM when subjected to stretch. Cytokines such as interleukin-13 and tumor necrosis factor-α promote a more contractile ASM phenotype. The composition and increased stiffness of the matrix in which ASM is embedded promotes a more proliferative and pro-inflammatory ASM phenotype, but the expected dedifferentiation and loss of contractility have not been shown. Airway epithelium may drive ASM proliferation and/or molecular remodeling in ways that may lead to AHR. In conclusion, AHR is likely multifactorial in origin, reflecting the plasticity of ASM properties in the inflammatory environment of the asthmatic airway.", "keywords": [ "Airway hyperresponsiveness", "smooth muscle", "Airway epithelium", "asthma" ], "content": "Introduction\n\nFor decades, the excessive responses of the asthmatic airway to bronchoconstrictive stimuli have been recognized in association with asthma. Standardization of bronchial provocation testing with histamine initially and methacholine subsequently led to quantification of airway responsiveness (AHR) and ultimately its incorporation into the definition of asthma. Although loosely associated with the severity of asthma, the quantification of AHR in the pulmonary function laboratory provides a measure of the probability that the person studied suffers from some of the symptoms attributable to asthma. Despite its limited clinical usefulness, the assessment of AHR to challenge with inhaled methacholine triggered by stimuli such as allergens has been the principal surrogate for asthma in attempts to model the disease in animals. The environment-host interactions leading to AHR are multiple and the explanation for AHR is likely multifactorial. It is intuitively obvious that airway smooth muscle (ASM) is at the center of the problem, since it is the principal actor in airway narrowing. However, the extent to which the milieu in which it functions is responsible for excessive shortening of ASM or altered properties of ASM confer the excess responsiveness remains unclear1. In addition, the expanded role of ASM in the biology of the airway has emerged as the synthetic capacity of ASM has been elucidated. This commentary reviews some of the proposed explanations for the occurrence of AHR with a particular focus on the ASM.\n\n\nMechanical determinants of AHR\n\nAirway narrowing results from the effects of ASM contraction and has been thoroughly analyzed from the standpoint of its mechanical determinants. A static analysis of a model of the airway tree as a tube which constricts in response to contraction of ASM embedded in the wall has provided useful insights into the determinants of airway resistance and AHR2–4. The theoretical importance of geometric factors such as increase in area of tissue between the ASM and the lumen, the mass of ASM, and the elastic recoil of the parenchyma surrounding the airway was demonstrated. The exquisite sensitivity of airway responsiveness to lung volume in vivo5,6 supports the idea that lung elastic recoil is crucial in opposing the capacity of ASM to narrow the airways. However, a static force-balance analysis is incomplete. Interaction of contracting ASM with stresses imposed on the airway wall by breathing has demonstrated substantial effects on ASM behavior (Figure 1).\n\nAirway narrowing is favored by enhanced contractility of airway smooth muscle (ASM) and by an increase in mass of ASM. Potent inhibitors of airway narrowing are lung elastic recoil, airway-parenchymal interdependence, and oscillatory stretches of the ASM caused by breathing movements and intermittent deep breaths.\n\nThe lack of a bronchodilating effect of a deep breath in asthma has been extensively documented and has prompted its further exploration as a fundamental characteristic of the asthmatic airway7. The imposition of a tidal breathing pattern that precludes any deep breath during the inhalation of the bronchoconstrictor methacholine augments the degree of induced bronchoconstriction in non-asthmatic subjects8. The reason for the failure of asthmatics to respond to a deep breath with bronchodilation remains uncertain. Stiffening of the asthmatic airway from remodeling9 is one potential reason, since the lengthening of the ASM will be less for any given change in airway transmural pressure imposed by a breath. Observations on intact human subjects have thus far not resolved the fundamental issue of the root cause of AHR. Perhaps further study of tissues from human lungs will help to clarify.\n\n\nContractile properties of ASM\n\nASM exhibits force-length characteristics reminiscent of skeletal muscle, although it retains the capacity to generate force over a much wider range and to shorten to much smaller lengths10. The range of shortening suffices for complete airway closure, and so effective mechanisms to limit shortening are required, as discussed above. A particularly remarkable property of ASM is its ability to readjust its molecular motors when length changes are sustained so that it regains the ability to generate force11. In other words, its force-length relationship is plastic and appears to be best explained by the addition or subtraction of myosin-containing contractile units in series to accommodate the new length12. Additionally, the induction of increased tone in ASM results in an increase in force generated in response to a further contractile stimulus13, suggesting that bronchoconstriction may beget bronchoconstriction. The maintenance of force by ASM may involve additional molecular mechanisms that affect actin-myosin interactions and potentially actin regulatory proteins such as calponin14 and caldesmon15. Cytoskeletal re-organization that may not involve the classical contractile apparatus of ASM may also affect force generation, a process that is sensitive to the inhibition of Rho kinase16.\n\nIn recent years, much attention has been paid to the interaction of superimposed length changes on ASM that is concomitantly stimulated to contract. In vitro cyclical stretching of pre-contracted ASM causes the ASM to lose much of its tension17. These oscillatory forces applied to the ASM are predicted to be equivalent to the stresses imposed by tidal breathing on the airways leading to a reduction of active force, described as fluidization. Whether tidal breathing exerts the predicted degrees of stretch of ASM in vivo that have been used in vitro has been questioned18. Some of the impact of stretching of ASM may be offset by changes in the contractile apparatus that counteract the dilating effects of the imposed length changes19. Chin and colleagues have found that tracheal smooth muscle harvested from asthmatic subjects shows a smaller reduction in force following imposed length changes compared to non-asthmatic tissues20. Another study that failed to confirm these findings may have studied asthmatics of lesser severity21. If severe asthma is associated with a resistance of contracted ASM to lose force with length change, then understanding this phenomenon will help us to understand the observed lack of bronchodilation in these subjects after a deep breath7,8. Currently, the softening of ASM is not completely explained but is attributed to disruption of either actin-myosin bridges or cytoskeletal proteins that contribute to the cellular stiffness. Fluidization is also sensitive to and enhanced by Rho kinase inhibition16. This pathway may in the future provide therapeutic possibilities for the reversal of severe bronchoconstriction as witnessed during asthma attacks, where reversal by usual bronchodilator therapy is not observed.\n\nIt seems inconceivable that the ASM does not have a central role in AHR. Rats that show innate hyperresponsiveness to contractile agonists in vivo also show ASM hypercontractility at the level of tissue and cells22,23. These findings appear to be explained by differences in the metabolism of inositol trisphosphate24, the principal mediator of calcium release within ASM cells. In contrast studies of the properties of ASM harvested from asthmatic subjects have, in general, failed to reveal hyperresponsiveness at the tissue level20,21, although peripheral airways have not been well studied. However, impaired relaxation has been observed and linked to β-adrenergic receptor uncoupling25 and greater degradation of 3’,5’-cyclic adenosine monophosphate by phosphodiesterase26. Ultrastructural studies of trachealis muscle have failed also to reveal differences between tissues harvested from asthmatic and non-asthmatic subjects27. Biopsied ASM shows changes in the expression of a range of contractile proteins, compatible with molecular remodeling of the ASM in asthma28,29. However, not all studies report similar findings30, although differences in the severity of the asthma may account for some of the discrepancies. In equine asthma (heaves), one of the few authentic models of asthma, the smooth muscle of the peripheral airways of the heaves-affected horses exhibited a greater unloaded shortening velocity (maximal velocity of contraction [Vmax]) compared to their own trachealis and compared to peripheral ASM of control horses31. This supports the need for further studies of the more relevant intrapulmonary airways.\n\nIt is easily conceivable that an increase in ASM force generation, rather than an increase in the Vmax, may lead to AHR. An increase in ASM force is likely to override the impedance to airway narrowing resulting from lung elastic recoil and airway-parenchymal interdependence. The force, however, is difficult to quantify, as it depends on the stress generated by the ASM and the total amount of muscle and thus must be normalized by the cross-sectional area (stress) of ASM. Studies have so far shown no differences in stress between asthmatic and control airways20,21, but if the total amount of ASM is increased then the total force will increase too. This will require further mechanical investigations at the scale of the whole airway, preferably addressing the peripheral airways. The mechanism by which an increase in Vmax may lead to AHR is by counteracting the relaxing effect of tidal breaths1,32 by rapid recontraction. Studies using high-resolution computed tomography have shown that the airways from asthmatic subjects dilate upon stretching but that they quickly get back to their initial diameter33.\n\nAlterations in ASM in response to the inflammatory environment represent another mechanism to account for AHR and one that may be distinct from innate differences in ASM properties (Figure 2). Several cytokines involved in many cases of asthma such as interleukin (IL)-13 and tumor necrosis factor-α (TNF-α) alter the properties of ASM through effects on calcium signaling34. IL-13 evokes a calcium signal in murine ASM cells and causes their contraction35. Human ASM cells do not show this calcium spike following treatment with IL-13 but have enhanced calcium responses to stimulation by histamine36. A series of studies has linked this finding to an upregulation of the bifunctional enzyme CD3837, which synthesizes cyclic adenosine diphosphate (ADP) ribose, the mediator of calcium release, via the ryanodine receptor. TNF-α appears to enhance calcium signaling by similar mechanisms38. IL-13 additionally increases the sensitivity of the molecular motors to calcium by stimulating Rho kinase39. IL-13 has also been found to reduce the relaxant response to ASM cells to β-adrenergic stimulation40 and is the Th2 cytokine that has been most closely linked to AHR. The sensitization of ASM by immunoglobulin E (IgE) also leads to enhanced contractility and again by mechanisms involving IL-13 but by its autocrine effects on ASM41. IL-13 also triggers oscillatory calcium currents42, which may lead to transcriptional effects, and downregulates a regulator of G-proteins, RGS2, leading to AHR in mice43.\n\nAirway smooth muscle (ASM) function is modulated by cytokines such as interleukin (IL)-13 and tumor necrosis factor (TNF)-α that increase intracellular calcium release in response to agonists such as histamine. ASM is triggered to proliferate in vivo by release of epidermal growth factor receptor (EGFR) ligands. The stiffness and composition of the matrix change ASM phenotype and may promote pro-inflammatory properties.\n\nContact of ASM with immune cells has also been shown to enhance the contractility of smooth muscle. Several studies have implicated T lymphocytes in the phenomenon41,44. The effect of T cells on ASM contractility has been linked to autocrine production of IL-5 and IL-1β. Incubation of rat tracheal rings with CD4+ T cells leads to an increased Vmax along with alterations in contractile protein expression45. These changes necessitate contact between the muscle and the T cells, suggesting that intercellular cell adhesion mechanisms are involved. Mast cells within the ASM bundles express IL-1346 and may also be a cellular mechanism by which contractility of ASM is altered in asthma.\n\n\nProliferative/secretory properties of ASM\n\nASM may exist in different states, contractile or secretory/proliferative, and ASM cells harvested from asthmatic subjects differ in these states from those of non-asthmatic subjects, retaining characteristics across multiple passages in cell culture. Asthmatic ASM proliferates more readily, a characteristic that has been attributed to increased mitochondrial mass that is in turn related to calcium handling47. This abnormality, which is conserved in vitro, is presumably nonetheless an induced property. Alterations in the transcriptional regulation of secreted molecules such as CXCL8 and synthesis of enzymes such as cycolooxygenase-2 by ASM cells are now being reported48–50 and may provide clues to the altered states of asthmatic ASM. The profile of matrix protein production by asthmatic and non-asthmatic ASM differs51 with the possibility of altering the milieu in terms of its potential to support an inflammatory environment or to affect other aspects of ASM phenotype.\n\n\nChanges in ASM mass\n\nFor almost 100 years, it has been noted that ASM is present in increased quantities in the airways of asthmatics. Formal morphometric studies in the 1960s documented the changes in a quantitative manner52. Given the abovementioned issues of the balance of force offsetting ASM shortening, the increase in mass has the potential to account for AHR3. Such a conclusion hinges on whether the contractile properties of the ASM are unaltered by its growth. In vitro, such states are generally reciprocally regulated so that proliferation of the ASM is anticipated to result in a reduction in contractile proteins53. Whether such a phenomenon occurs in vivo awaits demonstration. One such study suggested changes in proliferating trachealis54, but a subsequent study found no decline in myosin heavy chain despite significant proliferation of cells within the tissue studied55. In human asthmatic tissues, contractile cells show a substantial variation in the intensity of staining for smooth muscle-specific α-actin, suggesting cells with a range of contractile properties exist in vivo56. Those staining poorly for α-actin were not well incorporated into smooth muscle bundles but were arranged loosely and without any obvious relationship to existing ASM architecture. These cells may have been myofibroblasts, long argued to be precursors of ASM. The orientation of ASM within the airway wall may have a profound influence on the degree of airway narrowing that follows from the ASM contraction57. There is currently no evidence that addresses this latter issue.\n\nIncrease in ASM mass results predominantly from an increase in the number of cells58. In some cases, hypertrophy contributes to the change. Several studies have observed proliferating cells among the ASM bundles, suggesting measureable turnover of the ASM tissue56,59. A corollary is that remodeling of the ASM is dynamic and therefore potentially reversible, although no studies in humans have reported such a phenomenon. Only modest changes in AHR result from corticosteroid treatment of asthma, suggesting that remodeling may be irreversible, assuming it accounts for AHR in the first place. However, in equine asthma, partial reversibility of increased ASM mass has been shown with antigen avoidance or corticosteroid treatment60.\n\nIf the mass of ASM is increasing, what factors are responsible for the increase? Many studies have been performed on animal models showing that ASM remodeling can be inhibited by attenuating airway inflammation, a finding that is hardly surprising, although purely mechanical factors related to compression of the airway epithelium by bronchoconstriction may release potent growth factors such as heparin-binding epidermal growth factor-like growth factor (HB-EGF)61. Other studies also implicate the airway epithelium as a potential source of modulators of ASM properties. The cysteinyl leukotriene LTD4 releases HB-EGF from airway epithelial cells62, as does IL-1363. HB-EGF will not only cause ASM proliferation but also promote goblet cell differentiation. The EGF receptor and cysteinyl leukotrienes have both been implicated in ASM and goblet cell differentiation induced by repeated allergen challenge in murine models64. Airway epithelial cells in co-culture exert a pro-proliferative effect on ASM cells, in part mediated by IL-6, IL-8, and monocyte chemoattractant protein-165. ASM from asthmatic subjects proliferates more readily than non-asthmatic ASM in response to the stimulation of airway epithelium by house dust mite and involves the activation of protease-activated receptor-2, epithelial synthesis of cysteinyl-leukotriene, and increased expression of the cysteinyl-leukotriene-1 receptor66.\n\nOver-expression of platelet-derived growth factor by airway epithelial cells may cause ASM growth in mice and is elevated in the bronchoalveolar lavage of mice after allergen challenges67, but whether it is involved in ASM remodeling caused by allergen is still unclear. Translation of the above findings to human asthma is yet to be done.\n\n\nMatrix and ASM properties\n\nMatrix composition and stiffness both influence the programming of cells, including those of the ASM. ASM cells cultured on collagen-conjugated polyacrylamide hydrogels of varying elastic moduli increase cellular secretion of vascular endothelial growth factor (VEGF)68. Stiff gels stimulate cell proliferation, reduce VEGF secretion, and reduce agonist-induced calcium responses of ASM cells68. However, in vitro studies demonstrate that susceptibility of ASM growth to inhibition by corticosteroids is affected also by its culture upon collagen I, whereas inhibiting the binding to collagen via α2β integrin restores the sensitivity to corticosteroids69. In vivo, the amount of collagen III and laminin in the ASM correlated with reversibility of the forced expiratory volume in 1 second (FEV1)70.\n\n\nConclusions\n\nAHR in the asthmatic subject remains poorly explained. However, the behavior of ASM in the dynamic circumstances of bronchoconstriction in vivo may prove to be the cause of its excessive narrowing if the failure of usual mechanisms limiting bronchoconstriction occurs in asthma. Accelerated rates of contraction, impaired relaxation, and blunted relaxant responses to tidal breathing and deep inspirations are among the potential characteristics that might lead to greater airway narrowing. Stiffening of ASM because of cytoskeletal re-organization could conceivably fix the airway in a narrowed state, relatively refractory to relaxant agonists, as is observed in acute asthma exacerbations. Reprogramming of ASM seems to occur, or perhaps selective overgrowth of ASM with a greater capacity for proliferation and pro-inflammatory effects occurs. Such reprogramming might be expected to lead to ASM that is less contractile but has not been shown. The role that altered airway mechanics or epithelial effects plays in the ASM program may require further exploration.\n\n\nAbbreviations\n\nASM, airway smooth muscle; AHR, airway hyperresponsiveness; EGFR, epidermal growth factor receptor; HB-EGF, heparin-binding epidermal growth factor-like growth factor; IL, interleukin; TNF-α, tumor necrosis factor-α; Vmax, maximal velocity of contraction.", "appendix": "Competing interests\n\n\n\nThe author(s) declare that they have no competing interests.\n\n\nGrant information\n\nJames G. Martin* and Anne-Marie Lauzon are supported by grants from the Canadian Institutes of Health Research and the Richard and Edith Strauss Canada Foundation.\n\n\nReferences\n\nSolway J, Fredberg JJ: Perhaps airway smooth muscle dysfunction contributes to asthmatic bronchial hyperresponsiveness after all. Am J Respir Cell Mol Biol. 1997; 17(2): 144–6. PubMed Abstract \| Publisher Full Text\n\nMoreno RH, Hogg JC, Paré PD: Mechanics of airway narrowing. 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Eur Respir J. 2008; 32(5): 1213–23. PubMed Abstract \| Publisher Full Text\n\nMalavia NK, Raub CB, Mahon SB, et al.: Airway epithelium stimulates smooth muscle proliferation. Am J Respir Cell Mol Biol. 2009; 41(3): 297–304. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nTrian T, Allard B, Dupin I, et al.: House dust mites induce proliferation of severe asthmatic smooth muscle cells via an epithelium-dependent pathway. Am J Respir Crit Care Med. 2015; 191(5): 538–46. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nHirota JA, Ask K, Farkas L, et al.: In vivo role of platelet-derived growth factor-BB in airway smooth muscle proliferation in mouse lung. Am J Respir Cell Mol Biol. 2011; 45(3): 566–72. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nShkumatov A, Thompson M, Choi KM, et al.: Matrix stiffness-modulated proliferation and secretory function of the airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol. 2015; 308(11): L1125–35. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBonacci JV, Schuliga M, Harris T, et al.: Collagen impairs glucocorticoid actions in airway smooth muscle through integrin signalling. Br J Pharmacol. 2006; 149(4): 365–73. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nYick CY, Ferreira DS, Annoni R, et al.: Extracellular matrix in airway smooth muscle is associated with dynamics of airway function in asthma. Allergy. 2012; 67(4): 552–9. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation" }	[ { "id": "12816", "date": "09 Mar 2016", "name": "Christina Pabelick", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12817", "date": "09 Mar 2016", "name": "Chun Seow", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-306
https://f1000research.com/articles/5-300/v1	08 Mar 16	{ "type": "Review", "title": "Advances in the prevention, management, and treatment of community-acquired pneumonia", "authors": [ "Mathias W. Pletz", "Gernot G. Rohde", "Tobias Welte", "Martin Kolditz", "Sebastian Ott", "Gernot G. Rohde", "Tobias Welte", "Martin Kolditz", "Sebastian Ott" ], "abstract": "Community-acquired pneumonia (CAP) is the infectious disease with the highest number of deaths worldwide. Nevertheless, its importance is often underestimated. Large cohorts of patients with CAP have been established worldwide and improved our knowledge about CAP by far. Therefore, current guidelines are much more evidence-based than ever before. This article discusses recent major studies and concepts on CAP such as the role of biomarkers, appropriate risk stratification to identify patients in need of hospitalisation or intensive care, appropriate empiric antibiotic therapy (including the impact of macrolide combination therapy and antibiotic stewardship), and CAP prevention with novel influenza and pneumococcal vaccines.", "keywords": [ "community-acquired pneumonia", "Streptococcus pneumoniae", "pulmonary infection", "CAP" ], "content": "Introduction and epidemiology\n\nCommunity-acquired pneumonia (CAP) remains a burden in the modern world. The annual incidence ranges from between 2.7 and 10 per 1000 persons and has not changed much during the last few decades1. In Germany, around 250,000 patients are hospitalised because of CAP each year, and it is expected that twice as many patients with CAP are managed in primary care2. The incidence shows a U-shaped distribution from very young to very old age3. CAP is still one of the most important reasons for premature death, particularly in developing countries and in children4.\n\nCAP is an infectious disease of the lung parenchyma and adjacent organs. Respiratory bacteria constitute the major group of causative organisms. However, there must be some caution, as in most studies in more than 50% of the cases no pathogen could be identified5. Streptococcus pneumoniae is the pathogen most frequently identified throughout all studies and settings (outpatients, inpatients, and intensive care unit [ICU] patients with CAP)5–7. Haemophilus influenzae (HI) is also frequently detected in outpatients (13%) but much less in hospitalised patients (6% to 7%)5. A possible reason is the high prevalence of HI in patients with chronic obstructive pulmonary disease (COPD)8, patients who are at high risk of developing CAP9. Another important group of pathogens is respiratory viruses, particularly in outpatients but also inpatients, much less in ICU patients5. New detection techniques such as multiplex-polymerase chain reaction allow better insight into the relevant spectrum of viruses involved10. This fast-track diagnostic also allows us to put pandemic virus emergence into perspective, as shown during the H1N1 pandemic, in which respiratory syncytial virus and human metapneumovirus were the most prevalent viruses and only pandemic influenza virus A/H1N1 (2009), and not seasonal influenza virus, was detected11. Outside pandemics, seasonal influenza viruses cause yearly increases in CAP incidence and lead to increased mortality in patients co-infected with bacterial pathogens12,13. Another relevant group are the so-called “atypical” bacteria. Mycoplasma pneumonia is frequent in young patients with CAP (7% to 12%) and usually shows a benign course14,15. Chlamydophila pneumoniae historically has been reported to be a frequent pathogen mainly on the basis of serological assays. However, more recent research using molecular techniques found significantly lower detections rates (21% versus 3%, respectively)15. Legionella pneumophila has been identified as a causative pathogen with different frequencies5,16,17. It also occurs in outpatients, who commonly show a more favorable course of disease than inpatients16.\n\n\nBiomarkers\n\nHistorically, pro-inflammatory biomarkers such as leucocyte count and C-reactive protein (CRP) are widely used in CAP. In most patients with CAP, these markers are elevated and show the highest levels in bacterial CAP, followed by atypical CAP and viral CAP18. In mixed (bacterial + viral) CAP, CRP levels seem to be highest but the predictive value is low19. However, an individual prediction of CAP aetiology is not possible18. Also, procalcitonin (PCT), which shows a very fast response during infections, is not able to predict aetiology of CAP18. However, PCT levels on admission can support the identification of severe outcomes of CAP and add to the prognostic properties of clinical risk score20. It has a higher prognostic accuracy compared with CRP and leucocyte count21. This has been recently confirmed22. Interestingly, PCT levels can provide independent identification of patients at low risk of death within CRB-65 (confusion of new onset, respiratory rate of at least 30 breaths per minute, blood pressure of less than 90 mmHg systolic or diastolic blood pressure of 60 mmHg or less, and age of at least 65 years) classes21. Importantly, antibiotic pre-treatment has to be taken into account, as it influences the prognostic properties23.\n\nA very recent study showed that the diagnostic accuracies of CRP and PCT are insufficient to confirm CAP if the diagnosis is established by using a gold standard that includes thoracic computed tomography (CT) scan24. However, in primary care, the addition of CRP (optimal cutoff of more than 30 mg/L) improved the diagnosis of CAP in patients with typical signs and symptoms, whereas PCT did not add clinically relevant information25.\n\nIn the most comprehensive study on the prognostic properties of new CAP biomarkers (including mid-regional pro-adrenomedullin [MR-proADM], mid-regional pro-atrial natriuretic peptide [MR-proANP], pro-arginine-vasopressin [copeptin], proendothelin-1 [CT-proET-1], PCT, CRP, white blood cell count, and CRB-65 score), MR-proADM, a cardiovascular biomarker, showed the best individual and a combination of CRB-65 with MR-proADM showed the best overall prognostic performance26,27.\n\nHyponatremia is common on admission among patients with CAP and was independently associated with mortality. The combination of sodium and pro-vasopressin and pro-ANP levels achieved the highest prediction of mortality in a recent analysis28.\n\nA very simple but powerful biomarker is admission blood glucose (Glc). Already mildly elevated Glc levels were significantly associated with an increased short-term mortality odds ratio (OR) of 1.56 with further increases at higher Glc levels. Therefore, acute hyperglycaemia may identify patients in need of intensified care to reduce the risk of death from CAP29. Potentially along comparable mechanisms and independently of clinical scores and inflammatory biomarkers, increased serum cortisol levels have been associated with mortality in CAP30. On the other hand, admission hypoglycaemia has also been associated with increased short- and long-term mortality31, underlining the value of Glc as a useful biomarker.\n\n\nRisk stratification\n\nCAP shows a highly variable disease course; published mortality rates vary between less than 1% and more than 40% according to treatment setting, disease severity, age, and comorbidities5. Whereas mortality and complication rates remain low in non-severe CAP managed in the community32, hospitalised CAP is associated with a high risk of respiratory failure or sepsis-related organ dysfunction. CAP mortality of hospitalised patients in Germany continues to be about 13%, rising to more than 35% in patients with need of mechanical ventilation2. Although treatment restrictions and functional status of multi-morbid patients might influence such mortality figures33, even after conservative estimates excluding all patients residing in nursing homes or being bedridden before the CAP event, mortality remains more than 7%, which matches mortality rates of other recognised medical emergency conditions such as ST-elevation myocardial infarction34. This tremendous prognostic spectrum requires timely and adequate risk stratification as a crucial first step in CAP management to determine level of care and treatment intensity. From a clinical perspective, risk stratification is divided into two major components: (1) identification of patients with a low complication risk in the outpatient setting suitable for ambulatory treatment and (2) identification of patients with high risk for acute organ failure necessitating early intensified management and monitoring interventions in the emergency department.\n\nTo assist clinical risk assessment, different score systems have been recommended by guidelines. Most established are the pneumonia severity index (PSI) score35, consisting of 20 clinical, laboratory, and radiographic variables, and the CRB-65 or CURB criteria, which include only four parameters32,36. They proved to be comparable tools for mortality prediction37,38, but the CRB-65 score is preferred in the outpatient setting, as it is easy to calculate and works without laboratory parameters. However, its sensitivity in elderly and multi-morbid patients is suboptimal, and poor functional status has been shown as a major independent mortality predictor for these patients in a recent analysis33. Additionally, pre-existing chronic comorbidities have been independently associated with adverse prognosis in CAP2,35,39–42. In particular, acute cardiac complications are of major prognostic impact41,42. Therefore, the absence of (potentially) decompensating comorbidities is another precondition for ambulatory treatment. Recent studies identified poor oxygenation as an independent predictor for complications and mortality despite a low CRB-65 score43–45. Accordingly, a score adding poor oxygenation (saturation of less than 90%) and potentially decompensating comorbidities to the CRB-65 criteria recently has been introduced and validated in a large study from the CAPNETZ cohort, showing superior low-risk prediction compared with the original criteria39,40,46. The resulting risk evaluation to identify patients suitable for ambulatory treatment in the outpatient setting is depicted in Table 1.\n\nAcute pulmonary or extra-pulmonary organ dysfunction due to sepsis or decompensating (especially cardiovascular) comorbidities determines early prognosis in CAP41,47–49. Organ dysfunction risk is highest within the first 3 days after hospitalisation2,41,48,50,51. Efforts have been made to characterise a subgroup with an “emergency presentation” of CAP defined by a high risk of early clinical deterioration in order to target intensified management interventions to patients with a high potential of prognosis improvement47,52. A management-based risk stratification to allocate interventions like monitoring of organ functions, guideline-concordant management of severe sepsis, and early parenteral antimicrobial combination treatment has been suggested45,53. Obviously, patients presenting with immediate need for mechanical ventilation or vasopressor treatment are identified as medical emergencies. However, a recent analysis from the CAPNETZ cohort showed that prognosis is poorest in patients not presenting with these “major criteria” but deteriorating in the short course of the disease47. Independent predictors for early deterioration were vital sign abnormalities at presentation, highlighting the need for careful clinical patient evaluation. Recent studies have shown that the American Thoracic Society/Infectious Diseases Society of America (ATS/IDSA) minor criteria, which represent parameters of acute organ dysfunction, improve high-risk prediction47,54–56. Their accuracy to predict organ replacement therapy (optimal cutoff > two criteria) has been confirmed in a meta-analysis56, and outcome improvement after management intensification guided by these criteria in the emergency room has been demonstrated in an interventional trial57. Therefore, clinical high-risk identification should focus on acute respiratory or extra-pulmonary sepsis- or comorbidity-associated organ dysfunction, and careful clinical evaluation should be complemented by regular assessment of vital sign abnormalities and the minor criteria as shown in Table 2.\n\nATS/IDSA, American Thoracic Society/Infectious Diseases Society of America.\n\n\nAdjuvant treatment\n\nDespite adequate antimicrobial therapy, approximately 10% of all patients with CAP will not survive their current pulmonary infection, and mortality rates for severe CAP levelled off at 20% to 30% for the last few decades, especially if treatment failure occurs58. An overwhelming immunological response is assumed to be one of the key pathophysiological mechanisms behind the stagnating mortality rates. Keeping in mind the empty pipeline for new antimicrobial agents and the increasing threat of antibiotic resistance, adjuvant therapeutic strategies beyond just killing the causative microbes are urgently needed and are now the focus of research. Medicinal modulation or suppression of the immunologic host response (e.g. by the application of corticosteroids) might be a valid approach. This concept has been successfully implemented in the treatment of different infectious conditions such as bacterial meningitis and septic shock.\n\nFollowing the first positive corticosteroid trial in CAP by Confalonieri et al.59, conflicting results mainly from small clinical trials and meta-analysis questioned the potential benefit, and systematic reviews asked for larger randomised trials60–62. In 2015, two randomised controlled trials (RCTs) of adequate sample size were published, readopting the concept of adjuvant corticosteroid treatment in CAP63,64. Both studies appear to be supportive of corticosteroid administration, but before changing clinical practice, it is worth taking a closer look at their findings.\n\nBlum et al. randomly assigned hospitalised patients with CAP (n = 785; 70% PSI III-V) to receive an adjuvant treatment with 50 mg prednisolone daily in addition to standard care63. The primary endpoint of this study was time to clinical stability and it was reached 1.4 days earlier in the treatment group (3.0 versus 4.4 days, hazard ratio [HR] 1.33, P <0.001). Additionally, steroid administration facilitated an earlier switch to oral sequence therapy (4.0 versus 5.0 days, P = 0.011), shortened the length of hospitalisation (6.0 versus 7.0 days, HR 1.19, P = 0.012), and reduced the incidence of pneumonia-associated complications like acute respiratory distress syndrome (ARDS) and empyema (OR 0.46, 95% confidence interval [CI] 0.22 to 0.98, P = 0.05). These effects were independent of PSI score at admission, initial levels of CRP, and underlying comorbidities such as COPD. Unfortunately, the study was not designed to address mortality, the most important clinical outcome, and the number of side effects (hyperglycemia) was significantly higher in the treatment group (19% versus 11%, OR 1.96, P = 0.001). Furthermore, overall mortality was comparably low (3.4%) and only few patients required ICU treatment (4.8%).\n\nThe second study, by Torres et al., included 120 CAP patients with high serum levels of inflammatory markers (CRP >150 mg/L)64. The patients were randomly assigned to receive 0.5 mg/kg methylprednisolone given twice a day for 5 days or placebo. The primary endpoint was occurrence of treatment failure, a composite endpoint including early failure (progression to septic shock, the need for mechanical ventilation, or death within 72 hours after admission) and late failure (radiographic progression, persisting respiratory failure, progression to septic shock, the need for mechanical ventilation, or death within 72 to 120 hours after admission). The authors found significantly less treatment failure in the treatment group (13% versus 31%, P = 0.02) and by trend a lower mortality. Again, the incidence of hyperglycemia was higher, but not statistically significant (18% versus 12%, P = 0.34). Although the results were positive at first sight, a critical interpretation is recommended. The composite endpoint “treatment failure” allows for misinterpretations, especially when considering that the only significant differences between the groups were found in “late failure” due to “radiographic progression” and “late-onset septic shock”. “Radiographic progression” alone does not necessarily reflect clinical failure; it needs to be accompanied by clinical instability to be indicative of treatment failure, and “late-onset septic shock” is not always attributable to CAP.\n\nIn conclusion, there is growing evidence of some beneficial effects of steroid treatment in terms of faster resolution of clinical signs and symptoms and prevention of CAP-associated complications, but so far the impact on mortality cannot be judged sufficiently. This was confirmed by a recent meta-analysis indicating that steroids had no significant impact on mortality (relative risk [RR] 0.72, 95% CI 0.43 to 1.21), even in severe CAP (RR 0.72, 95% CI 0.43 to 1.21), but may prevent the development of ARDS (RR 0.21, 95% CI 0.08 to 0.59) and reduce the lengths of hospital and ICU stay and the time to clinical stability65. Hopefully, the results of ongoing trials will help to elucidate the future role of adjuvant corticosteroid treatment in the management of CAP66,67. Nevertheless, it is important to notice that supportive steroid treatment in patients with influenza CAP is associated with increased mortality68. Therefore, steroids should not be administered to patients with proven influenza, except for asthmatics and COPD patients who may require systemic steroids for the treatment of bronchial obstruction, even in the context of influenza69.\n\nRecently, it has been reported that statins may have immunomodulatory and anti-inflammatory properties and that their current intake may have favourable effects on the course of respiratory infections, including pneumonia70,71. However, most of the knowledge is derived from retrospective case-control studies including patients with already-established statin intake, and only few prospective RCTs are available.\n\nIn one large, population-based, case-control study (n >100,000), the recent use of statins significantly reduced the risk of mortality from pneumonia (adjusted OR 0.47) but had an effect neither on the incidence of non-severe pneumonia in the study population nor on the need for hospitalisation due to pneumonia72. Two additional case-control studies found a 22% reduced risk of pneumonia in patients with current exposure to statins73,74. However, the protective effect was not shown in other studies, possibly indicating a “healthy user” bias75,76.\n\nOne recent randomised, double-blind, placebo-controlled trial addressed the effects of de novo statin use on CAP outcome and blood levels of inflammatory cytokines77. In this study, the use of 20 mg simvastatin once daily for 4 days since hospital admission did not reduce the time to clinical stability and the levels of inflammatory cytokines in patients hospitalised with CAP. However, it is worth mentioning that the authors failed to achieve their recruitment target for determining the effect of de novo statin use on their clinical endpoint (time to clinical stability). Another randomised, double-blind, placebo-controlled trial focused on the effects of simvastatin (60 mg daily) on day 28 mortality in patients with ventilator-associated pneumonia78. This study was prematurely stopped for futility after interim analyses with 300 patients enrolled. There was no difference in the primary endpoint (day 28 mortality) between groups (statin group 21.2% versus placebo group 15.2%, P = 0.1).\n\nIn conclusion, there is some evidence indicating that an established statin exposure may reduce the risk of pneumonia and have beneficial effects on the clinical course, but the results are conflicting and the findings of first RCTs on de novo statin use are discouraging. Therefore, the use of statins for primary prophylaxis or as adjuvant pneumonia therapy may not be recommended at present.\n\n\nAntibiotic treatment\n\nMost major guidelines suggest an empiric treatment stratified according to severity of disease79,80. Outpatients are treated orally with penicillins, macrolides, tetracyclines, or fluoroquinolones with anti-pneumococcal activity (i.e. moxifloxacin or levofloxacin). Oral cephalosporins have been linked to increased treatment failure (OR 2.86, 95% CI 1.56 to 5.27), probably due to the unfavourable bioavailability plus—compared with intravenous administration—low licensed dosages81. Recent EUCAST (European Committee on Antimicrobial Susceptibility Testing) guidelines state (e.g. for HI) that cefuroxime breakpoints apply only to high-dose treatment (i.e. 1.5 g three times daily).\n\nAs outlined above, the proportion of “atypical” pathogens has been probably overestimated in the past, particularly due to the low specificity of serology for detection of Chlamydophila pneumoniae82.\n\nSince coverage of atypical bacteria by macrolides, fluoroquinolones, or tetracyclines seems to be expandable in mild cases treated as outpatients—most guidelines recommend oral penicillins or aminopenicillins (longer half life time, higher bioavailability, and better activity against HI than penicillin V) to cover pneumococci—empiric combination treatment of inpatients with beta-lactam plus macrolide remains an issue of debate79,80.\n\nBesides covering “atypical” pathogens, macrolides are supposed to attenuate the inflammatory response by decreasing expression of pro-inflammatory cytokines and consecutive neutrophil recruitment to lung parenchyma. A retrospective study revealed a clinical advantage of macrolides even in patients with macrolide-resistant pneumococcal pneumonia. However, recently, the cardiotoxicity of macrolides has been linked to a slightly increased mortality. A meta-analysis found that erythromycin carries the greatest risk of QT prolongation and torsades de pointes from all macrolides, followed by clarithromycin and azithromycin83. A large Danish cohort study estimated 37 cardiac deaths in 1 million treatments with clarithromycin84, with an increased risk particularly in women. Whereas the Svanstrom study addressed younger adults and not patients with CAP, the study by Ray et al. showed a higher risk of sudden death with azithromycin compared with amoxicillin85. The study by Schembri et al. is currently the only one that looked specifically at protocol-defined CAP and showed an increased risk of long-term cardiac events86. However, Mortensen et al. showed that the benefit of azithromycin in reducing CAP mortality outweighed the risk of cardiotoxicity87.\n\nA 2014 meta-analysis comprising four prospective cohort studies and 12 retrospective cohort studies (n = 42,942) found a decreased mortality for macrolide/beta-lactams versus beta-lactam monotherapy88. However, randomised studies were not available.\n\nFinally, in 2015, two randomised studies addressing this question were published. A cluster-randomised non-inferiority study from The Netherlands compared beta-lactam monotherapy, beta-lactam/macrolide, and fluoroquinolone for empiric treatment of CAP89. A Swiss open-label, multi-centre, non-inferiority, randomised trial compared cefuroxime or amoxicillin/clavulanic acid with or without clarithromycin90. The Swiss study could not prove non-inferiority for beta-lactam monotherapy regarding the proportion of patients reaching clinical stability on day 7, even after exclusion of patients with a positive urine legionella antigen test result. In contrast, the Dutch study found that beta-lactam monotherapy was non-inferior to strategies with a beta-lactam-macrolide combination or fluoroquinolone monotherapy with regard to 90-day mortality. The macrolide used in the Dutch study was erythromycin91, which has a higher cardiotoxicity than azithromycin or clarithromycin83. The Swiss study showed that, in particular, patients with atypical pathogens (mostly Mycoplasma pneumoniae) and patients with a higher severity profited from the macrolide combination. This supports the obligated empiric beta-lactam/macrolide combination treatment for at least all CAP patients admitted to the ICU, a strategy suggested by most major guidelines80,81,91.\n\n\nAntibiotic stewardship\n\nAntibiotic stewardship has become an important strategy to fight the antibiotic resistance crisis. How is antibiotic stewardship implemented in CAP treatment? First of all, pneumonia has to be differentiated from non-pneumonia entities (e.g. bronchitis and acute exacerbation of COPD) in patients presenting with lower respiratory tract infections. This requires a standard chest X-ray, which is frequently not available in the outpatient setting. Several studies have shown that using a PCT is a useful biomarker to decide for or against empiric antibiotics in inpatients and outpatients presenting with lower respiratory tract infections. PCT-guided strategies have decreased (unnecessary) antibiotic prescriptions by 30% to 50% without impairing clinical outcome92,93. Similar data are available in primary care for CRP, which—in contrast to PCT—is available as a point-of-care test94.\n\nOther approaches used a clinical score to predict CAP in outpatients presenting with acute respiratory tract infection in order to identify patients who should be prescribed antibiotics95.\n\nAnother strategy to decrease antibiotic consumption without harming the patient is to shorten antibiotic treatment. A recent prospective before-and-after intervention study from Scotland describes the implementation of a simple CRB-65-based algorithm for duration of treatment (i.e. CAP: 5 days of antibiotics for mild and 7 for moderate/severe cases. Acute exacerbation of COPD: 5 days of antibiotics and no antibiotics at all in patients without an increase in sputum purulence)96. This algorithm was enforced by automatic stop dates and pharmacist feedback to prescribers and resulted in significant reductions of antibiotic consumption and in antibiotic side effects without increasing mortality or length of stay.\n\nVaccines are available against pneumococci and influenza virus, the most frequent bacterial and viral causes of CAP, respectively. Bacterial-viral co-infections are associated with increased mortality, and synergistic effects have been shown for combined vaccination12.\n\nThe standard influenza vaccine is the trivalent split vaccine, containing two influenza A and one influenza B strains, which are annually selected by the World Health Organization. Within the last few years, efforts have been made to improve acceptance, coverage of the vaccine, and particularly its efficacy in the elderly. A central problem of the influenza vaccine is that the elderly, who are at increased risk, exhibit an inferior response to the vaccine because of immunosenescence. Intradermal vaccination aims to stimulate more antigen-presenting cells, which are found in higher concentration in the dermis than in the subcutis or the muscle. Virions mimic natural viral cell entry, and adjuvants aim to recruit more antigen-presenting cells. High-dose vaccines use four times the amount of antigen. Whereas studies have shown that most of these approaches increase antibody titres, only high-dose vaccines were tested in a study with a clinically relevant endpoint and showed an increased prevention of laboratory-confirmed influenza cases97. Other strategies try to improve the influenza vaccine coverage. In contrast to influenza A, influenza B does not undergo antigenic shift and therefore does not cause pandemics. However, as a result of accumulated point mutations, influenza B split into two lines (Yamagata and Victoria) about 30 to 40 years ago98. Historically, only one influenza B line was included in the trivalent split vaccine. Therefore, the coverage of the trivalent split vaccine has depended on the accurate prediction of the dominating B line in the particular season. Recently, quadrivalent influenza vaccines that include both influenza B lines have been made available for clinical use99.\n\nPneumococcal vaccination of adults is a current issue of debate, since both a 23-valent polysaccharide vaccine and a 13-valent conjugate vaccine are licensed for use in adults. Meta-analyses have shown that the polysaccharide vaccine prevents pneumococcal bacteraemia with an efficacy of about 75%. However, the majority of pneumococcal pneumonia is non-bacteraemic, and it remains controversial whether this vaccine is protective against non-invasive pneumococcal pneumonia100. To date, only one RCT in Japanese nursing home residents showed a clear reduction of pneumococcal pneumonia101, whereas several other studies and a respective meta-analysis revealed no effect100. In a recent meta-analysis comparing the four available RCTs on 23-valent polysaccharide pneumococcal vaccine (PPV23) efficacy against CAP, the study by Maruyama et al.101 has been identified as an outlier, contributing statistically significant heterogeneity to this analysis102. Recently, a large Dutch placebo-controlled multi-centre study showed that the 13-valent conjugate vaccine prevented non-invasive pneumococcal pneumonia due to vaccine serotypes with an efficacy of 45%103. The coverage of 13-valent pneumococcal conjugate vaccine (PCV13) in adults is supposed to decrease because of herd protection effects of the PCV13 infant vaccination program that has led to a substantial decrease of the 13 vaccine serotypes in countries with such a program. Nevertheless, it remains unclear whether the herd protection effects on invasive pneumococcal disease seen after implementing PCV7 can be extrapolated to the additional six serotypes of PCV13 or non-invasive pneumococcal pneumonia or both. Recent data from Sweden, the US, and Germany suggest that there is only minor or no herd protection for serotype 3, one of the most frequent serotypes causing pneumonia in adults104–106. Considering these data, the Advisory Committee on Immunization Practices has suggested a sequential vaccination (PCV13 followed by PPV23 after 6 to 12 months) for all adults older than 65. Within the next few years, intensive surveillance on serotype distribution in pneumococcal pneumonia is needed in order to estimate the extension of herd protection and to evaluate the use of PCV13 vaccination in adults.\n\n\nImplications for clinical practice\n\nCAP is the infectious disease with the highest number of deaths worldwide. Nevertheless, the importance of this disease is often underestimated. It is diagnosed too late, severity scoring is not appropriate, so that patients are too seldom admitted to intermediate care or ICUs, and antibiotic therapy is often not in accordance with guidelines. Large cohorts of patients with CAP have been established worldwide and vastly improved our knowledge about CAP. Therefore, current guidelines are much more evidence based than ever before. The challenge for the future is to implement current knowledge into clinical practice to reduce the number of CAP cases (by vaccination) and the number of deaths (by adequate diagnostics and treatment). National and international societies should establish CAP audits to oversee the management of CAP and to give clinicians feedback about their daily clinical practice.", "appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\nAll of the following reported fees and grants were received outside the submitted work: MWP and TW received fees for lectures/advisory board participation from AstraZeneca, Basilea, Bayer, GSK, MSD, Novartis, and Pfizer. TW received fees for research grants from Bayer, Novartis, and Pfizer. MWP received fees for research grants from Infectopharm, Biotest, and Pfizer. GR has received consulting/lecture fees/honoraria from Novartis, Takeda, Astra-Zeneca, Chiesi, Grünenthal, GlaxoSmithKline, and Pfizer. SO has received consulting/lecture fees/honoraria from Bayer, GlaxoSmithKline, and Pfizer. MK reports grants and lecture fees from Pfizer and lecture fees from Thermo-Fisher, Gilead, GlaxoSmithKline, Novartis, and Böhringer-Ingelheim.\n\n\nGrant information\n\nMP was supported by a grant from the German Ministry of Education and Research (grant number 01KI1204).\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nSchnoor M, Hedicke J, Dalhoff K, et al.: Approaches to estimate the population-based incidence of community acquired pneumonia. J Infect. 2007; 55(3): 233–9. PubMed Abstract \| Publisher Full Text\n\nEwig S, Birkner N, Strauss R, et al.: New perspectives on community-acquired pneumonia in 388 406 patients. Results from a nationwide mandatory performance measurement programme in healthcare quality. Thorax. 2009; 64(12): 1062–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJansen AG, Rodenburg GD, de Greeff SC, et al.: Invasive pneumococcal disease in the Netherlands: Syndromes, outcome and potential vaccine benefits. Vaccine. 2009; 27(17): 2394–401. PubMed Abstract \| Publisher Full Text\n\nGBD 2013 Mortality and Causes of Death Collaborators: Global, regional, and national age-sex specific all-cause and cause-specific mortality for 240 causes of death, 1990-2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet. 2015; 385(9963): 117–71. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBraeken D, Franssen F, Schütte H, et al.: Increased Severity and Mortality of CAP in COPD: Results from the German Competence Network, CAPNETZ. J COPD F. 2014; 2(2): 131–40. Publisher Full Text\n\nBierbaum S, Königsfeld N, Besazza N, et al.: Performance of a novel microarray multiplex PCR for the detection of 23 respiratory pathogens (SYMP-ARI study). Eur J Clin Microbiol Infect Dis. 2012; 31(10): 2851–61. PubMed Abstract \| Publisher Full Text\n\nBierbaum S, Forster J, Berner R, et al.: Detection of respiratory viruses using a multiplex real-time PCR assay in Germany, 2009/10. Arch Virol. 2014; 159(4): 669–76. PubMed Abstract \| Publisher Full Text\n\nvon Baum H, Schweiger B, Welte T, et al.: How deadly is seasonal influenza-associated pneumonia? The German Competence Network for Community-Acquired Pneumonia. Eur Respir J. 2011; 37(5): 1151–7. PubMed Abstract \| Publisher Full Text\n\nJohansson N, Kalin M, Tiveljung-Lindell A, et al.: Etiology of community-acquired pneumonia: increased microbiological yield with new diagnostic methods. Clin Infect Dis. 2010; 50(2): 202–9. PubMed Abstract \| Publisher Full Text\n\nvon Baum H, Welte T, Marre R, et al.: Mycoplasma pneumoniae pneumonia revisited within the German Competence Network for Community-acquired pneumonia (CAPNETZ). BMC Infect Dis. 2009; 9: 62. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDumke R, Schnee C, Pletz MW, et al.: Mycoplasma pneumoniae and Chlamydia spp. infection in community-acquired pneumonia, Germany, 2011–2012. Emerging Infect Dis. 2015; 21(3): 426–34. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvon Baum H, Ewig S, Marre R, et al.: Community-acquired Legionella pneumonia: new insights from the German competence network for community acquired pneumonia. Clin Infect Dis. 2008; 46(9): 1356–64. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nArancibia F, Cortes CP, Valdés M, et al.: Importance of Legionella pneumophila in the etiology of severe community-acquired pneumonia in Santiago, Chile. Chest. 2014; 145(2): 290–6. PubMed Abstract \| Publisher Full Text\n\nKrüger S, Ewig S, Papassotiriou J, et al.: Inflammatory parameters predict etiologic patterns but do not allow for individual prediction of etiology in patients with CAP: results from the German competence network CAPNETZ. Respir Res. 2009; 10(1): 65. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBello S, Mincholé E, Fandos S, et al.: Inflammatory response in mixed viral-bacterial community-acquired pneumonia. BMC Pulm Med. 2014; 14: 123. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nSchuetz P, Suter-Widmer I, Chaudri A, et al.: Prognostic value of procalcitonin in community-acquired pneumonia. Eur Respir J. 2011; 37(2): 384–92. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nKrüger S, Ewig S, Marre R, et al.: Procalcitonin predicts patients at low risk of death from community-acquired pneumonia across all CRB-65 classes. Eur Respir J. 2008; 31(2): 349–55. PubMed Abstract \| Publisher Full Text\n\nZhydkov A, Christ-Crain M, Thomann R, et al.: Utility of procalcitonin, C-reactive protein and white blood cells alone and in combination for the prediction of clinical outcomes in community-acquired pneumonia. Clin Chem Lab Med. 2015; 53(4): 559–66. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nKrüger S, Ewig S, Kunde J, et al.: Assessment of inflammatory markers in patients with community-acquired pneumonia--influence of antimicrobial pre-treatment: results from the German competence network CAPNETZ. Clin Chim Acta. 2010; 411(23–24): 1929–34. PubMed Abstract \| Publisher Full Text\n\nLe Bel J, Hausfater P, Chenevier-Gobeaux C, et al.: Diagnostic accuracy of C-reactive protein and procalcitonin in suspected community-acquired pneumonia adults visiting emergency department and having a systematic thoracic CT scan. Crit Care. 2015; 19: 366. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nvan Vugt SF, Broekhuizen BD, Lammens C, et al.: Use of serum C reactive protein and procalcitonin concentrations in addition to symptoms and signs to predict pneumonia in patients presenting to primary care with acute cough: diagnostic study. BMJ. 2013; 346: f2450. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nKrüger S, Ewig S, Giersdorf S, et al.: Cardiovascular and inflammatory biomarkers to predict short- and long-term survival in community-acquired pneumonia: Results from the German Competence Network, CAPNETZ. Am J Respir Crit Care Med. 2010; 182(11): 1426–34. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nEspaña PP, Capelastegui A, Mar C, et al.: Performance of pro-adrenomedullin for identifying adverse outcomes in community-acquired pneumonia. J Infect. 2015; 70(5): 457–66. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nKrüger S, Ewig S, Giersdorf S, et al.: Dysnatremia, vasopressin, atrial natriuretic peptide and mortality in patients with community-acquired pneumonia: results from the german competence network CAPNETZ. Respir Med. 2014; 108(11): 1696–705. PubMed Abstract \| Publisher Full Text\n\nLepper PM, Ott S, Nüesch E, et al.: Serum glucose levels for predicting death in patients admitted to hospital for community acquired pneumonia: prospective cohort study. BMJ. 2012; 344: e3397. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nKolditz M, Höffken G, Martus P, et al.: Serum cortisol predicts death and critical disease independently of CRB-65 score in community-acquired pneumonia: a prospective observational cohort study. BMC Infect Dis. 2012; 12: 90. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nGamble JM, Eurich DT, Marrie TJ, et al.: Admission hypoglycemia and increased mortality in patients hospitalized with pneumonia. Am J Med. 2010; 123(6): 556.e11–6. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nBauer TT, Ewig S, Marre R, et al.: CRB-65 predicts death from community-acquired pneumonia. J Intern Med. 2006; 260(1): 93–101. PubMed Abstract \| Publisher Full Text\n\nEwig S, Bauer T, Richter K, et al.: Prediction of in-hospital death from community-acquired pneumonia by varying CRB-age groups. Eur Respir J. 2013; 41(4): 917–22. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nBauer TT, Welte T, Strauss R, et al.: Why do nonsurvivors from community-acquired pneumonia not receive ventilatory support? Lung. 2013; 191(4): 417–24. PubMed Abstract \| Publisher Full Text\n\nFine MJ, Auble TE, Yealy DM, et al.: A prediction rule to identify low-risk patients with community-acquired pneumonia. N Engl J Med. 1997; 336(4): 243–50. PubMed Abstract \| Publisher Full Text\n\nLim WS, van der Eerden MM, Laing R, et al.: Defining community acquired pneumonia severity on presentation to hospital: an international derivation and validation study. Thorax. 2003; 58(5): 377–82. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLoke YK, Kwok CS, Niruban A, et al.: Value of severity scales in predicting mortality from community-acquired pneumonia: systematic review and meta-analysis. Thorax. 2010; 65(10): 884–90. PubMed Abstract \| Publisher Full Text\n\nChalmers JD, Singanayagam A, Akram AR, et al.: Severity assessment tools for predicting mortality in hospitalised patients with community-acquired pneumonia. Systematic review and meta-analysis. Thorax. 2010; 65(10): 878–83. PubMed Abstract \| Publisher Full Text\n\nKolditz M, Ewig S, Schütte H, et al.: Assessment of oxygenation and comorbidities improves outcome prediction in patients with community-acquired pneumonia with a low CRB-65 score. J Intern Med. 2015; 278(2): 193–202. PubMed Abstract \| Publisher Full Text\n\nDwyer R, Hedlund J, Henriques-Normark B, et al.: Improvement of CRB-65 as a prognostic tool in adult patients with community-acquired pneumonia. BMJ Open Respir Res. 2014; 1(1): e000038. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nCorrales-Medina VF, Musher DM, Wells GA, et al.: Cardiac complications in patients with community-acquired pneumonia: incidence, timing, risk factors, and association with short-term mortality. Circulation. 2012; 125(6): 773–81. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nAliberti S, Ramirez J, Cosentini R, et al.: Acute myocardial infarction versus other cardiovascular events in community-acquired pneumonia. ERJ Open Res. 2015; 1(1): 00020-2015-20-2015. Publisher Full Text \| F1000 Recommendation\n\nEl-Solh AA, Alhajhusain A, Abou Jaoude P, et al.: Validity of severity scores in hospitalized patients with nursing home-acquired pneumonia. Chest. 2010; 138(6): 1371–6. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nMajumdar SR, Eurich DT, Gamble JM, et al.: Oxygen saturations less than 92% are associated with major adverse events in outpatients with pneumonia: a population-based cohort study. Clin Infect Dis. 2011; 52(3): 325–31. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nChoudhury G, Chalmers JD, Mandal P, et al.: Physician judgement is a crucial adjunct to pneumonia severity scores in low-risk patients. Eur Respir J. 2011; 38(3): 643–8. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nDwyer R, Hedlund J, Darenberg J, et al.: Improvement of CRB-65 as a prognostic scoring system in adult patients with bacteraemic pneumococcal pneumonia. Scand J Infect Dis. 2011; 43(6–7): 448–55. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nKolditz M, Ewig S, Klapdor B, et al.: Community-acquired pneumonia as medical emergency: predictors of early deterioration. Thorax. 2015; 70(6): 551–8. PubMed Abstract \| Publisher Full Text\n\nAliberti S, Amir A, Peyrani P, et al.: Incidence, etiology, timing, and risk factors for clinical failure in hospitalized patients with community-acquired pneumonia. Chest. 2008; 134(5): 955–62. PubMed Abstract \| Publisher Full Text\n\nAliberti S, Brambilla AM, Chalmers JD, et al.: Phenotyping community-acquired pneumonia according to the presence of acute respiratory failure and severe sepsis. Respir Res. 2014; 15(1): 27. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nDremsizov T, Clermont G, Kellum JA, et al.: Severe sepsis in community-acquired pneumonia: when does it happen, and do systemic inflammatory response syndrome criteria help predict course? Chest. 2006; 129(4): 968–78. PubMed Abstract \| Publisher Full Text\n\nPhua J, Ngerng WJ, Lim TK: The impact of a delay in intensive care unit admission for community-acquired pneumonia. Eur Respir J. 2010; 36(4): 826–33. PubMed Abstract \| Publisher Full Text\n\nEwig S, Torres A: Community-acquired pneumonia as an emergency: time for an aggressive intervention to lower mortality. Eur Respir J. 2011; 38(2): 253–60. PubMed Abstract \| Publisher Full Text\n\nKolditz M, Ewig S, Höffken G: Management-based risk prediction in community-acquired pneumonia by scores and biomarkers. Eur Respir J. 2013; 41(4): 974–84. PubMed Abstract \| Publisher Full Text\n\nChalmers JD, Mandal P, Singanayagam A, et al.: Severity assessment tools to guide ICU admission in community-acquired pneumonia: systematic review and meta-analysis. Intensive Care Med. 2011; 37(9): 1409–20. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nChalmers JD, Taylor JK, Mandal P, et al.: Validation of the Infectious Diseases Society of America/American Thoratic Society minor criteria for intensive care unit admission in community-acquired pneumonia patients without major criteria or contraindications to intensive care unit care. Clin Infect Dis. 2011; 53(6): 503–11. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nSalih W, Schembri S, Chalmers JD: Simplification of the IDSA/ATS criteria for severe CAP using meta-analysis and observational data. Eur Respir J. 2014; 43(3): 842–51. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nLim HF, Phua J, Mukhopadhyay A, et al.: IDSA/ATS minor criteria aid pre-intensive care unit resuscitation in severe community-acquired pneumonia. Eur Respir J. 2014; 43(3): 852–62. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nOtt SR, Hauptmeier BM, Ernen C, et al.: Treatment failure in pneumonia: impact of antibiotic treatment and cost analysis. Eur Respir J. 2012; 39(3): 611–8. PubMed Abstract \| Publisher Full Text\n\nConfalonieri M, Urbino R, Potena A, et al.: Hydrocortisone infusion for severe community-acquired pneumonia: a preliminary randomized study. Am J Respir Crit Care Med. 2005; 171(3): 242–8. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nSnijders D, Daniels JM, de Graaff CS, et al.: Efficacy of corticosteroids in community-acquired pneumonia: a randomized double-blinded clinical trial. Am J Respir Crit Care Med. 2010; 181(9): 975–82. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nFernández-Serrano S, Dorca J, Garcia-Vidal C, et al.: Effect of corticosteroids on the clinical course of community-acquired pneumonia: a randomized controlled trial. Crit Care. 2011; 15(2): R96. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMeijvis SC, Hardeman H, Remmelts HH, et al.: Dexamethasone and length of hospital stay in patients with community-acquired pneumonia: a randomised, double-blind, placebo-controlled trial. Lancet. 2011; 377(9782): 2023–30. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nBlum CA, Nigro N, Briel M, et al.: Adjunct prednisone therapy for patients with community-acquired pneumonia: a multicentre, double-blind, randomised, placebo-controlled trial. Lancet. 2015; 385(9977): 1511–8. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nTorres A, Sibila O, Ferrer M, et al.: Effect of corticosteroids on treatment failure among hospitalized patients with severe community-acquired pneumonia and high inflammatory response: a randomized clinical trial. JAMA. 2015; 313(7): 677–86. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nWan YD, Sun TW, Liu ZQ, et al.: Efficacy and Safety of Corticosteroids for Community-Acquired Pneumonia: A Systematic Review and Meta-Analysis. Chest. 2016; 149(1): 209–19. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nADRENAL trial. ClinicalTrials.gov. NCT01448109. Reference Source\n\nESCAPe trial. ClinicalTrials.gov. NCT01283009. Reference Source\n\nRodrigo C, Leonardi-Bee J, Nguyen-Van-Tam JS, et al.: Effect of corticosteroid therapy on influenza-related mortality: a systematic review and meta-analysis. J Infect Dis. 2015; 212(2): 183–94. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nMyles P, Nguyen-Van-Tam JS, Semple MG, et al.: Differences between asthmatics and nonasthmatics hospitalised with influenza A infection. Eur Respir J. 2013; 41(4): 824–31. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nViasus D, Garcia-Vidal C, Gudiol F, et al.: Statins for community-acquired pneumonia: current state of the science. Eur J Clin Microbiol Infect Dis. 2010; 29(2): 143–52. PubMed Abstract \| Publisher Full Text\n\nChalmers JD, Short PM, Mandal P, et al.: Statins in community acquired pneumonia: Evidence from experimental and clinical studies. Respir Med. 2010; 104(8): 1081–91. PubMed Abstract \| Publisher Full Text\n\nSchlienger RG, Fedson DS, Jick SS, et al.: Statins and the risk of pneumonia: a population-based, nested case-control study. Pharmacotherapy. 2007; 27(3): 325–32. PubMed Abstract \| Publisher Full Text\n\nMyles PR, Hubbard RB, McKeever TM, et al.: Risk of community-acquired pneumonia and the use of statins, ace inhibitors and gastric acid suppressants: a population-based case-control study. Pharmacoepidemiol Drug Saf. 2009; 18(4): 269–75. PubMed Abstract \| Publisher Full Text\n\nVinogradova Y, Coupland C, Hippisley-Cox J: Risk of pneumonia in patients taking statins: population-based nested case-control study. Br J Gen Pract. 2011; 61(592): e742–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDublin S, Jackson ML, Nelson JC, et al.: Statin use and risk of community acquired pneumonia in older people: population based case-control study. BMJ. 2009; 338: b2137. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFleming DM, Verlander NQ, Elliot AJ, et al.: An assessment of the effect of statin use on the incidence of acute respiratory infections in England during winters 1998–1999 to 2005–2006. Epidemiol Infect. 2010; 138(9): 1281–8. PubMed Abstract \| Publisher Full Text\n\nViasus D, Garcia-Vidal C, Simonetti AF, et al.: The effect of simvastatin on inflammatory cytokines in community-acquired pneumonia: a randomised, double-blind, placebo-controlled trial. BMJ Open. 2015; 5(1): e006251. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nPapazian L, Roch A, Charles PE, et al.: Effect of statin therapy on mortality in patients with ventilator-associated pneumonia: a randomized clinical trial. JAMA. 2013; 310(16): 1692–700. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nHöffken G, Lorenz J, Kern W, et al.: Guidelines of the Paul-Ehrlich-Society of Chemotherapy, the German Respiratory Diseases Society, the German Infectious Diseases Society and of the Competence Network CAPNETZ for the Management of Lower Respiratory Tract Infections and Community-acquired Pneumonia. Pneumologie. 2010; 64(3): 149–54. PubMed Abstract \| Publisher Full Text\n\nWoodhead M, Blasi F, Ewig S, et al.: Guidelines for the management of adult lower respiratory tract infections--full version. Clin Microbiol Infect. 2011; 17(Suppl 6): E1–59. PubMed Abstract \| Publisher Full Text\n\nCreutz P, Kothe H, Braun M, et al.: Failure of Ambulatory Treatment in Cap Patients Leading to Subsequent Hospitalization and its Association to Risk Factors. J Pulmon Resp Med. 2013; 03(1). Publisher Full Text\n\nWellinghausen N, Straube E, Freidank H, et al.: Low prevalence of Chlamydia pneumoniae in adults with community-acquired pneumonia. Int J Med Microbiol. 2006; 296(7): 485–91. PubMed Abstract \| Publisher Full Text\n\nGuo D, Cai Y, Chai D, et al.: The cardiotoxicity of macrolides: a systematic review. Pharmazie. 2010; 65(9): 631–40. PubMed Abstract\n\nSvanström H, Pasternak B, Hviid A: Use of clarithromycin and roxithromycin and risk of cardiac death: cohort study. BMJ. 2014; 349: g4930. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRay WA, Murray KT, Hall K, et al.: Azithromycin and the risk of cardiovascular death. N Engl J Med. 2012; 366(20): 1881–90. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nSchembri S, Williamson PA, Short PM, et al.: Cardiovascular events after clarithromycin use in lower respiratory tract infections: analysis of two prospective cohort studies. BMJ. 2013; 346: f1235. PubMed Abstract \| Publisher Full Text\n\nMortensen EM, Halm EA, Pugh MJ, et al.: Association of azithromycin with mortality and cardiovascular events among older patients hospitalized with pneumonia. JAMA. 2014; 311(21): 2199–208. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNie W, Li B, Xiu Q: β-Lactam/macrolide dual therapy versus β-lactam monotherapy for the treatment of community-acquired pneumonia in adults: a systematic review and meta-analysis. J Antimicrob Chemother. 2014; 69(6): 1441–6. PubMed Abstract \| Publisher Full Text\n\nPostma DF, van Werkhoven CH, van Elden LJ, et al.: Antibiotic treatment strategies for community-acquired pneumonia in adults. N Engl J Med. 2015; 372(14): 1312–23. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nGarin N, Genné D, Carballo S, et al.: β-Lactam monotherapy vs β-lactam-macrolide combination treatment in moderately severe community-acquired pneumonia: a randomized noninferiority trial. JAMA Intern Med. 2014; 174(12): 1894–901. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nSchouten JA, Prins JM, Bonten MJ, et al.: Revised SWAB guidelines for antimicrobial therapy of community-acquired pneumonia. Neth J Med. 2005; 63(8): 323–35. PubMed Abstract\n\nBurkhardt O, Ewig S, Haagen U, et al.: Procalcitonin guidance and reduction of antibiotic use in acute respiratory tract infection. Eur Respir J. 2010; 36(3): 601–7. PubMed Abstract \| Publisher Full Text\n\nSchuetz P, Briel M, Christ-Crain M, et al.: Procalcitonin to guide initiation and duration of antibiotic treatment in acute respiratory infections: an individual patient data meta-analysis. Clin Infect Dis. 2012; 55(5): 651–62. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nCooke J, Butler C, Hopstaken R, et al.: Narrative review of primary care point-of-care testing (POCT) and antibacterial use in respiratory tract infection (RTI). BMJ Open Respir Res. 2015; 2(1): e000086. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLittle P, Stuart B, Moore M, et al.: Amoxicillin for acute lower-respiratory-tract infection in primary care when pneumonia is not suspected: a 12-country, randomised, placebo-controlled trial. Lancet Infect Dis. 2013; 13(2): 123–9. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nMurray C, Shaw A, Lloyd M, et al.: A multidisciplinary intervention to reduce antibiotic duration in lower respiratory tract infections. J Antimicrob Chemother. 2014; 69(2): 515–8. PubMed Abstract \| Publisher Full Text\n\nDiazGranados CA, Dunning AJ, Kimmel M, et al.: Efficacy of high-dose versus standard-dose influenza vaccine in older adults. N Engl J Med. 2014; 371(7): 635–45. PubMed Abstract \| Publisher Full Text\n\nPletz MW, Welte T: Pneumococcal and influenza vaccination. In: Chalmers JD, Pletz MW, Aliberti S, editors. Community-Acquired Pneumonia. European Respiratory Society, 2014; 266–284. Reference Source\n\nCrépey P, de Boer PT, Postma MJ, et al.: Retrospective public health impact of a quadrivalent influenza vaccine in the United States. Influenza Other Respir Viruses. 2015; 9(Suppl 1): 39–46. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoberley S, Holden J, Tatham DP, et al.: Vaccines for preventing pneumococcal infection in adults. Cochrane Database Syst Rev. 2013; 1: CD000422. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nMaruyama T, Taguchi O, Niederman MS, et al.: Efficacy of 23-valent pneumococcal vaccine in preventing pneumonia and improving survival in nursing home residents: double blind, randomised and placebo controlled trial. BMJ. 2010; 340: c1004. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchiffner-Rohe J, Witt A, Hemmerling J, et al.: Efficacy of PPV23 in Preventing Pneumococcal Pneumonia in Adults at Increased Risk - A Systematic Review and Meta-Analysis. PLoS One. 2016; 11(1): e0146338. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nBonten MJ, Huijts SM, Bolkenbaas M, et al.: Polysaccharide conjugate vaccine against pneumococcal pneumonia in adults. N Engl J Med. 2015; 372(12): 1114–25. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nGalanis I, Lindstrand A, Darenberg J, et al.: Effects of PCV7 and PCV13 on invasive pneumococcal disease and carriage in Stockholm, Sweden. Eur Respir J. 2016; 47(3): pii: ERJ-01451-2015. PubMed Abstract \| Publisher Full Text\n\nRichter SS, Diekema DJ, Heilmann KP, et al.: Changes in pneumococcal serotypes and antimicrobial resistance after introduction of the 13-valent conjugate vaccine in the United States. Antimicrob Agents Chemother. 2014; 58(11): 6484–9. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nvan der Linden M, Falkenhorst G, Perniciaro S, et al.: Effects of Infant Pneumococcal Conjugate Vaccination on Serotype Distribution in Invasive Pneumococcal Disease among Children and Adults in Germany. PLoS One. 2015; 10(7): e0131494. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "12809", "date": "09 Mar 2016", "name": "James D. Chalmers", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12810", "date": "09 Mar 2016", "name": "Mats Kalin", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-300
https://f1000research.com/articles/5-299/v1	08 Mar 16	{ "type": "Case Report", "title": "Case Report: Rectal perforation during CT colonography", "authors": [ "Marianna Zukiwskyj", "Yasser Arafat", "Yasser Arafat" ], "abstract": "IntroductionComputer tomography colonoscopy (CTC) is an increasingly prevalent procedure for the investigation of colorectal symptoms, or as a component of colorectal cancer screening. It is considered a low risk procedure, however colonic perforation is a recognized significant complication. Case ReportWe report the case of an 81-year-old female patient who underwent CTC after failed optical colonoscopy as part of routine colorectal cancer screening. Perforation of the rectum with surrounding pararectal air was confirmed on CTC. The patient had minimal symptoms and was treated successful non-operatively with bowel rest and antibiotics. ConclusionPerforation sustained during CTC is an uncommon complication. The incidence of perforation during CTC is still lower than that during optical colonoscopy. In the absence of significant abdominal signs and symptoms, this rare complication may be successfully managed non-operatively.", "keywords": [ "Rectal perforation", "CT Colonography", "CTC" ], "content": "Introduction\n\nThe incidence of colorectal cancer worldwide has been reported as 9%1. It is the third most commonly diagnosed cancer worldwide1, and, excluding cutaneous cancers, is the mostly commonly diagnosed cancer in Australia2. National screening programmes are an important tool for the early detection of, and effective reduction in mortality from colorectal cancer2,3. Since its inception in 1994, computer tomography colonoscopy (CTC) has been increasingly utilized for both colorectal cancer screening and investigation of colorectal symptoms4. Indications for CTC include colorectal cancer screening, incomplete or failed optical colonoscopy, symptomatic or asymptomatic individuals who may have significant medical comorbidities considered high risk for optical colonoscopy. Advantages of CTC include minimal invasiveness, better patient tolerance, unlikely need for sedation, low incident of adverse events, and the occasional discovery of extra colonic pathology5,6. Colonic perforation is a significant albeit rare complication of CTC7.\n\n\nCase report\n\nAn 81-year-old Caucasian female underwent a CTC for colorectal cancer screening. She tested positive to a faecal occult blood stool test, and had experienced longstanding, infrequent, minimal and painless bright red rectal bleeding for a period of over twenty years. She gave no history of loss of weight, change in bowel habits, or family history of colorectal cancer. Past medical history was significant for atrial fibrillation requiring anticoagulation, diverticular disease and a hysterectomy 20 years prior with subsequent radiotherapy to the pelvis as histology had confirmed uterine cancer. In the last twenty years, four screening colonoscopies had been carried out, the last five years prior, and whilst clear of polyps, was significant for an asymptomatic narrowed segment of distal sigmoid which had to be traversed with a paediatric colonoscope. The patient underwent a CTC as, she had, earlier in the year, undergone a failed optical colonoscopy. CTC was carried out with manual air insufflation via a rectal soft tip Foley catheter until the patient felt slight discomfort, at which point a scout AP film was taken to ensure adequate bowel distension. It proceeded without complications. The official report noted a localized contained perforation demonstrated around the rectum (Figure 1, Figure 2). There was extensive diverticular disease demonstrated throughout the sigmoid colon, which was markedly narrowed throughout in keeping with stricture formation, which would be consistent with previous diverticulitis or radiation treatment (Figure 3). Further assessment of that region was difficult. The remainder of the large bowel had achieved excellent distension.\n\nThe Foley catheter tip is seen in rectum as well as localized peri-rectal air.\n\nThe Foley catheter is seen within the rectum as well as peri-rectal air.\n\nThe patient was admitted to the ward and commenced on prophylactic antibiotics (Tazocin 4.5g tds), and bowel rest. She complained of mild discomfort in the lower abdominal region, and had a soft abdomen with no evidence of peritonism. During the first two days of admission, she passed a small amount of blood clot per rectum. She was discharged on day five of admission, with no abdominal signs, having undergone an MRI which confirmed no peri-rectal collection and identified no obvious perforation. A further optical colonoscopy was attempted two months later, which failed to enter the sigmoid colon, citing severe diverticular disease as the reason. The patient currently remains asymptomatic.\n\n\nDiscussion\n\nCTC is widely considered as a minimally invasive technique with a low rate of adverse events. However, various complications arising from CTC have been reported in the literature. The most significant of these is colonic perforation. Since the inception of CTC, various publications have reported rates of perforation ranging from 0.009 to 0.1%4,6,7. This is in comparison to the rate of perforation at optical colonoscopy, which has been reported as ranging from 0.032% to 0.196%4, however is commonly quoted as 0.05% to 0.1%. The first meta-analysis on the rate of colonic perforated at CTC was conducted in 2014 by Bellini et al., in which an overall perforation rate of 0.04% was reported7. The majority of the perforations occurred in the sigmoid colon at 41%, with rectal perforations accounting for 22.2%7. When the perforation rate was adjusted for symptomatic or asymptomatic individuals (those for whom CTC was purely a screening procedure), the perforation rate of symptomatic patients was 0.08%, compared with asymptomatic patients at 0.02%, and the odds ratio was reported as 19.27. Various factors have been considered as increasing the likelihood of perforation. The use of rigid rectal catheters, CTC shortly after optical colonoscopy with biopsies, bowel containing inguinal herniae, a history of diverticular disease, CTC with manual insufflation of gas, and obstructing lesions have all been described as contributing to perforations in the literature4,7–9. However, given the rarity of perforations, the degree of contribution of each of the factors may vary amongst the current reviews.\n\nOnce a CTC perforation is confirmed, management may include operative and non-operative measures. The majority of reported CTC perforations have been managed non-operatively. Patients clinically suitable for non-operative management receive IV fluids, antibiotics and bowel rest. Bellini et al. reports that 68% of perforated patients were successful managed non-operatively7.\n\nIn this case, our patient, whilst asymptomatic of colorectal symptoms, did have several risk factors for perforation. There was a history of diverticular disease and of a narrowing in the region of the sigmoid, which did not require intervention at the time of her previous colonoscopy five years prior. Manual rather than automatic insufflation was employed. A soft tipped catheter was employed, and whilst this does lessen the risk of traumatic injury, it does not negate it, as the integrity of the rectal mucosa as well as technique of insertion are factors. As the diverticular disease and stricture extended to the distal sigmoid, it could be inferred that, in this case, the site more likely at risk of perforation would be rectal, rather than sigmoid. The most recent optical colonoscopy had been six months prior and so was unlikely to be a factor.\n\n\nConclusion\n\nColonoscopy is considered the gold standard for intraluminal evaluation of the colon in a variety of settings. CT colonography is an accepted alternative to optical colonoscopy in the event of failed endoscopic evaluation, as a screening procedure and in high risk candidates. The incidence of perforation at CTC is low. Bellini et al. reported fewer than 40 cases in their meta-analysis. As the majority of CTC perforations are managed non-operatively, the rate of CTC related surgical intervention was 0.008%7. Whilst the rate of perforation is accepted as lower than that of optical colonoscopy, the more significant advantage seems to be the much higher incidence of successful non-operative management of these patients7,10.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient.", "appendix": "Author contributions\n\n\n\nAll authors contributed to the content of this manuscript. MZ and YA 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\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nHaggar FA, Boushey RP: Colorectal cancer epidemiology: incidence, mortality, survival, and risk factors. Clin Colon Rectal Surg. 2009; 22(4): 191–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNational Health and Medical Research Council: Clinical practice guidelines for the prevention, early detection and management of colorectal cancer: a guide for general practitioners. Cancer Council Australia. 2008. Reference Source\n\nLevin B, Lieberman DA, McFarland B, et al.: Screening and surveillance for the early detection of colorectal cancer and adenomatous polyps, 2008: a joint guideline from the American Cancer Society, the US Multi-Society Task Force on Colorectal Cancer, and the American College of Radiology. Gastroenterology. 2008; 134(5): 1570–1595. PubMed Abstract \| Publisher Full Text\n\nIafrate F, Iussich G, Correale L, et al.: Adverse events of computed tomography colonography: an Italian National Survey. Dig Liver Dis. 2013; 45(8): 645–50. PubMed Abstract \| Publisher Full Text\n\nFlak B, Forster BB, Pezim ME: CT colonography: A new technique for colorectal cancer screening. BCMJ. 2008; 50(4): 206–11. Reference Source\n\nYee J, Weinstein S, Morgan T, et al.: Advances in CT Colonography for Colorectal Cancer Screening and Diagnosis. J Cancer. 2013; 4(3): 200–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBellini D, Rengo M, De Cecco CN, et al.: Perforation rate in CT colonography: a systematic review of the literature and meta-analysis. Eur Radiol. 2014; 24(7): 1487–96. PubMed Abstract \| Publisher Full Text\n\nBaccaro LM, Markelov A, Wilhelm J, et al.: Pneumoperitoneum after virtual colonoscopy: causes, risk factors, and management. Am Surg. 2014; 80(6): 549–54. PubMed Abstract\n\nAtalla MA, Rozen WM, Niewiadomski OD, et al.: Risk factors for colonic perforation after screening computed tomographic colonography: a multicentre analysis and review of the literature. J Med Screen. 2010; 17(2): 99–102. PubMed Abstract \| Publisher Full Text\n\nHamdani U, Naeem R, Haider F, et al.: Risk factors for colonoscopic perforation: a population-based study of 80118 cases. World J Gastroenterol. 2013; 19(23): 3596–601. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "12798", "date": "21 Mar 2016", "name": "Trevor Winter", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nComputer tomography colography (CTC) has been used since 1994 for colorectal screening and investigation of colorectal symptoms mainly in patients who have had incomplete optical colonoscopy, or who have significant medial comorbidities considered at high risk for optical colonoscopy. CTC is generally considered a minimally invasive procedure.\n\nThis paper is a useful addition to the literature, highlighting the low complication rate of CTC, with a perforation rate of 0.009% to 0.1%, compared to the complication rate of optical colonoscopy of 0.032% to 0.19%. The paper also highlights the factors associated with perforation as use of rigid rectal catheters, manual insufflation of gas CTC shortly after optical colonoscopy with biopsies, inguinal herniae, diverticular disease, and obstructing lesion. The authors comment that the majority of reported CTC perforations have been managed conservatively with IV fluids, antibiotics and bowel rest. Although colonoscopy is still considered the gold standard, CTC is a valuable alternative in patients with incomplete optical colonoscopy or are considered to be of high risk for optical colonoscopy due to medical comorbidities.", "responses": [] }, { "id": "12800", "date": "21 Mar 2016", "name": "Baljit Singh", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nA well written and interesting report. This highlights whilst this is a common procedure there is a small associated risk. However the authors suggest that in most cases this can be managed conservatively.", "responses": [] } ]	1	https://f1000research.com/articles/5-299
https://f1000research.com/articles/4-160/v1	23 Jun 15	{ "type": "Method Article", "title": "CRE: a cost effective and rapid approach for PCR-mediated concatenation of KRAS and EGFR exons", "authors": [ "Manoj P. Ramteke", "Kuldeep J Patel", "Mukul Godbole", "Maulik Vyas", "Kunal Karve", "Anuradha Choughule", "Kumar Prabhash", "Amit Dutt", "Manoj P. Ramteke", "Kuldeep J Patel", "Mukul Godbole", "Maulik Vyas", "Kunal Karve", "Anuradha Choughule" ], "abstract": "Molecular diagnostics has changed the way lung cancer patients are treated worldwide. Of several different testing methods available, PCR followed by directed sequencing and amplification refractory mutation system (ARMS) are the two most commonly used diagnostic methods worldwide to detect mutations at KRAS exon 2 and EGFR kinase domain exons 18-21 in lung cancer. Compared to ARMS, the PCR followed by directed sequencing approach is relatively inexpensive but more cumbersome to perform. Moreover, with a limiting amount of genomic DNA from clinical formalin-fixed, paraffin-embedded (FFPE) specimens or fine biopsies of lung tumors, multiple rounds of PCR and sequencing reactions often get challenging. Here, we report a novel and cost-effective single multiplex-PCR based method, CRE (for Co-amplification of five KRAS and EGFR exons), followed by concatenation of the PCR product as a single linear fragment for direct sequencing. CRE is a robust protocol that can be adapted for routine use in clinical diagnostics with reduced variability, cost and turnaround time requiring a minimal amount of template DNA extracted from FFPE or fresh frozen tumor samples. As a proof of principle, CRE is able to detect the activating EGFR L858R and T790M EGFR mutations in lung cancer cell line and primary tumors.", "keywords": [ "EGFR and KRAS mutation", "multiplex-PCR", "concatenation of PCR products", "Clinical diagnostics" ], "content": "Introduction\n\nThe growing significance of identifying EGFR and KRAS mutations in lung cancer using molecular diagnostic approaches underlines the emphasis on the use of personalized medical care by physicians to help design optimal therapeutic regimens (Lynch et al., 2004; Paez et al., 2004; Pao et al., 2004; Pao et al., 2005a; Pao et al., 2005b). While EGFR and KRAS mutations largely occur mutually exclusively in non-small cell lung cancer (NSCLC), and predict contrasting response rate to tyrosine-kinase inhibitors (TKI) (Chougule et al., 2013; Fukuoka et al., 2011; Ihle et al., 2012; Lynch et al., 2004; Mao et al., 2010; Mok et al., 2009), some recent studies, including ours, suggest co-occurrence of EGFR and KRAS mutations in the same patients, albeit at low frequency (Choughule et al., 2014; Li et al., 2014). These studies have direct implications for carrying out routine KRAS molecular testing along with EGFR mutations for precluding a patient with NSCLC from therapy with EGFR inhibitors, as approved for colorectal cancer (Lievre et al., 2006). Such information is especially important for lung cancer patients at an advanced-stage, who are not candidates for surgical intervention—wherein biopsy specimens obtained through fine-needle aspiration (FNA) may represent the only opportunity to obtain tissue material for diagnosis and molecular diagnostic analysis.\n\nEGFR mutations in NSCLC are characterized by approximately 39 unique mutations present across exons 18–21. Of these, most common are activating mutations, which account for approximately 90% of all EGFR mutations and are closely related to the efficacy of EGFR-TKIs. These activating mutations include point mutations G719S, T790M, L858R, and L861Q in exons 18, 20 and 21 respectively and in-frame deletions/insertions in exon 19 (Kosaka et al., 2004; Sharma et al., 2007 (review)). The most common mutations that result in an amino acid substitution at position 12 and 13 in KRAS are G12V and G13D (Choughule et al., 2014). Several screening and target based methods are currently in use for to infer the EGFR and KRAS hot spot mutations, viz; PCR-RFLP (Restriction fragment length polymorphism), Amplification Refractory Mutation System (ARMS), PCR-Invader, TaqMan PCR, allele specific qPCR, high resolution melting analysis and ultra-deep pyrosequencing, SNaPshot analysis and co-amplification at lower denaturation temperature (COLD)-PCR (Angulo et al., 2012; Borràs et al., 2011; Ellison et al., 2013; Santis et al., 2011; van Eijk et al., 2011; Zinsky et al., 2010). Of these, direct sequencing is the most commonly used method worldwide. However, a typical PCR reaction that precedes the sequencing step to amplify 4 EGFR and 1 KRAS exon(s) essentially consists of five rounds of independent PCR requiring separate aliquots of genomic DNA template for each reaction, followed by ten rounds of sequencing reactions. With a limited amount of genomic DNA from clinical FFPE specimens or fine biopsies of lung tumors, multiple rounds of PCR and sequencing reactions can often be challenging to perform.\n\nIn-frame concatenation or assembly of individually amplified exons from genomic DNA to generate a cDNA fragment has been described in earlier research, wherein the total number of PCR reactions corresponds to the number of exons to be concatenated (An et al., 2007; Fedchenko et al., 2013; Mitani et al., 2004; Tuohy & Groden, 1998). Here, we describe a novel methodology to co-amplify all four EGFR exons 18–21 along with KRAS exon 2 in a single multiplex PCR followed by directional or ordered concatenation of the products in the form of a single linear fragment. This concatenated product can be used to detect mutations by direct sequencing, at a much reduced cost and duration, and with a much smaller amount of template.\n\n\nMaterials and methods\n\nGenomic DNA was isolated from human NSCLC cell line NCI-H1975 and primary fresh frozen tumor tissue using QIAamp DNA blood mini kit (Qiagen). Genomic DNA from FFPE blocks was isolated using QIAamp DNA FFPE tissue kit (Qiagen) as per manufacturer’s instructions. DNA concentration was determined by absorbance at 280 nm (NanoDrop 2000, Thermo Scientific).\n\nPCR primers were designed for KRAS exon 2 and EGFR exons 18–21. Supplementary Table S1 represents all the primers used for PCR amplifications. With the exception of the OAD176 and OAD152 primers, all internal primers contain an additional overhang of 15 nucleotides, such that the tail sequence of forward and reverse primers of two subsequent exons are complementary to each other to allow ordered and directional concatenation of KRAS and EGFR exons. The full length concatenated product of 915 bases was amplified using OAD176 and OAD152 primers.\n\nMultiplex PCR (50µl per reaction) was carried out in a single tube by using multiplex PCR kit (Qiagen) containing either 10 ng of genomic DNA from the NSCLC cell line or fresh frozen primary tumor, or 50 ng of genomic DNA from FFPE blocks with 0.2 µM each of the five primer pairs using Applied Biosystems Veriti 96-well thermal cycler. PCR was carried out with initial hot-start denaturation at 95°C for 15 min, followed by 35 cycle of denaturation at 94°C for 30 seconds, annealing at 57°C for 90 seconds, polymerization at 72°C for 60 seconds, and final incubation for 30 min at 60°C. The multiplex PCR products were analyzed by agarose gel electrophoresis.\n\nFor concatenation of KRAS exon 2 and EGFR exons 18–21, 2 µl of multiplex PCR product was used as template in a 50 µl PCR reaction containing 0.2 µM of each OAD176 and OAD152 primers. PCR was carried out in a Applied Biosystems Veriti 96-well thermal cycler with an initial hot-start denaturation at 95°C for 15 min, followed by 35 cycle of denaturation at 94°C for 30 seconds, annealing at 57°C for 90 seconds, polymerization at 72°C for 60 seconds, and final incubation for 30 min at 60°C. Concatenated PCR product was analyzed by agarose gel electrophoresis. Sequencing of concatenated PCR products were performed by Sanger sequencing. Sequences were analyzed using Mutation Surveyor software V4.0.9 (Minton et al., 2011).\n\n\nResults\n\nCRE (Co-amplification of KRAS and EGFR exons) is a cost-effective multiplex-PCR based method followed by concatenation of the PCR product as a single fragment for direct sequencing (Figure 1). It is a robust methodology to determine the mutation status of KRAS and EGFR with reduced variability, cost and turnaround time, requiring a minimal amount of template DNA extracted from FFPE or fresh frozen tumor samples.\n\nThe flowchart represents the workflow for CRE methodology. KRAS and EGFR primers are shown along with complementary tail overhangs that prime with consecutive exons in an ordered manner. 2 µl PCR products, amplified with a cocktail of primers, as shown and described in Supplementary Table S1, for KRAS and EGFR exons in a single multiplex reaction is transferred to a fresh tube and concatenated in a separate reaction using OAD 176 and OAD 152 primers. The concatenated product obtained is a single product of 915 bp with all individual exons amplified from multiplex PCR ligated together in an ordered manner as a single fragment. 2x sequencing using the forward primer OAD 176 and reverse primer OAD 152 of the concatenated product is adequate to scan the mutation status across all the KRAS and EGFR exons.\n\n\nCRE-based KRAS-EGFR concatenation from fresh frozen primary tumors and tumor-derived cell lines\n\nFollowing CRE-based multiplex PCR of KRAS exon 2 and EGFR exons 18–21 with overlapping PCR bands (Figure 2A, lane 6), concatenation of the PCR product was performed with OAD176 and OAD152 primers using genomic DNA extracted from NCI-H1975 cells, a non-small-cell lung adenocarcinoma cell line. Concatenation PCR resulted in the enrichment of a concatenated product of about 915 base pairs (Figure 2B). This concatenated, gel purified PCR product of 915 base pairs was used for Sanger sequencing. Sequencing analysis of the concatenated PCR product confirmed concatenation as a single fragment (Figure 3) along with the presence of EGFR T790M and L585R mutations in NCI-H1975 cells (Supplementary Figure S1). A similar concatenation of a 915 bp single fragment was performed with genomic DNA extracted from fresh frozen tumor cells (Figure 2C).\n\nPanel A. PCR amplification of KRAS and EGFR exons using NCI-H1975 genomic DNA: Lane 1, KRAS exon 2 (151 bp) amplified with OAD176 and OAD177; Lane 2, EGFR exon 18 (209 bp) amplified with OAD 178 and OAD 144; Lane 3, EGFR exon 19 (178 bp) amplified with OAD 145 and OAD 146; Lane 4, EGFR exon 20 (246 bp) amplified with OAD 147 and OAD 150; Lane 5, EGFR exon 21 (251 bp) amplified with OAD 151 and OAD 152; Lane 6, Multiplex PCR of KRAS exon 2 and EGFR exons 18–21 with cocktail of primers used in Lanes 1–5.\n\nConcatenated KRAS and EGFR (CRE) product of ~915 bp amplified with OAD 176 and OAD 152 using multiplex PCR product as template derived from NCI-H1975 genomic DNA (shown in Panel B, Lane 2); derived from fresh frozen primary tumor genomic DNA (shown in Panel C, Lane 2); using tumor genomic DNA extracted from FFPE block (shown in Panel D, Lane 2).\n\nReverse complements of the forward sequencing reads of the 915 bp KRAS-EGFR concatenated product are displayed as generated by Mutation Surveyor V4.0.9. Panel A displays 15 nucleotide junction region flanked by KRAS exon 2 and EGFR exon 18 sequence; Panel B displays 15 nucleotide junction region flanked by EGFR exons 18 and 19; Panel C displays 15 nucleotide junction region flanked by EGFR exons 19 and 20; and displays 15 nucleotide junction region flanked by EGFR exons 20 and 21 is shown in Panel D.\n\n\nCRE-based KRAS-EGFR concatenation from paraffin-embedded clinical cancer specimens\n\nThe amount of genomic DNA obtained from FFPE tissue is always limiting and thus there is a substantial need to develop a technique with a limited amount of starting DNA as a template for mutation detection. CRE demonstrates the ability to co-amplify all five exons (KRAS exon 2 and EGFR exon 18–21) in a single multiplex PCR reaction with a limited amount of starting template DNA followed by the enrichment of concatenated product (Figure 2D) by concatenation PCR using first multiplex PCR product as a template. The concatenated product confirmed EGFR L858R mutation in the FFPE tissues (Supplementary Figure S2), as reported earlier (Choughule et al., 2014). Thus our CRE method can be routinely used for the mutational analysis of KRAS and EGFR genes.\n\n\nDiscussion\n\nCRE is a novel, simple and effective strategy to concatenate multiple amplicons obtained from a multiplex PCR, using primers with overlapping complementary overhangs. Compared to ARMS, and other genotyping technologies, CRE is relatively inexpensive with faster turnaround time involving lesser amount of template genomic DNA.\n\nUsing CRE, in vitro tandem reconstitution of KRAS exon 2 with EGFR exons 18–21 can be effectively performed to generate a concatenated single PCR product of 915 bp, as a template for sequencing. Most commercially-available allele-specific and genotyping technologies are restricted by their ability to probe only for eight out of the approximately 39 known commonly occurring EGFR and KRAS activating mutations. However, growing clinical data on the less common mutations are now emerging to fully inform their predictable outcomes on EGFR TKIs (Lohinai et al., 2015, Yang et al., 2012). Currently available methodologies, if extended to genotype all known 39 mutations would not only be cost-prohibitive but challenging to perform due to a limiting amount of template genomic DNA available from clinical cancer specimens that are mostly available in the form of formalin-fixed, paraffin-embedded (FFPE) tissue. While a directed sequencing approach –classical or next-generation sequencing (NGS) -based—can determine a whole spectrum of rare and co-occurring mutations in an individual, the question of template genomic DNA availability still remains. CRE circumvents the issue of a limiting amount of template genomic DNA with increased affordability by multiplexing PCR for all exons to a single reaction and concatenating the PCR product as a single fragment, thereby further reducing the cost of multiple sequencing reactions.\n\nIn this era of genome sequencing, applicability of the CRE strategy could be of immense significance to effectively reduce the cost and turnaround time taken to determine the mutational status across the whole KRAS exon 2 and EGFR kinase domain exons. As the limitation of the CRE strategy is defined by the sensitivity and resolution of the sequencing methodology adopted, concatenated EGFR and KRAS PCR products from multiple individuals—each tagged with unique bar code sequence—can be pooled and deep-sequenced using a NGS platform. The CRE strategy described here can reduce the labor and cost of performing individual PCR for all exons for each patient and effectively circumvent the noise due to variation in normalization for equimolar pooling of exons within the same sample at a resolution of single base.\n\n\nData availability\n\nF1000Research: Dataset 1. Raw gel electrophoresis images for Figure 2: Multiplex PCR amplification and concatenation of KRAS and EGFR exons generates CRE product, 10.5256/f1000research.6663.d50236\n\nF1000Research: Dataset 2. Sequencing traces for Figure 3: Full length sequencing of the CRE product, 10.5256/f1000research.6663.d50237\n\nF1000Research: Dataset 3. Sequencing traces for Figure S1: Detection of EGFR T790M and L858R mutations from NCI-H1975 CRE product, 10.5256/f1000research.6663.d50238\n\nF1000Research: Dataset 4. Sequencing trace for Figure S2: Detection of EGFR L858R mutation in a CRE product derived from FFPE primary tumor sample, 10.5256/f1000research.6663.d50239", "appendix": "Author contributions\n\n\n\nM.P.R. and K.J.P. contributed equally to this work. M.P.R., K.J.P, K.P. and A.D. conceived and designed the experiments. M.P.R., K.J.P, M.G., M.V., and K.K. performed the experiments. M.P.R., K.J.P. and A.D. analyzed the data. A.C. and K.P. contributed reagents/materials/analysis tools. M.P.R., K.J.P. and A.D. wrote the paper. All authors have seen and agreed to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nThe authors declared no competing interests.\n\n\nGrant information\n\nA.D. is supported by an Intermediate Fellowship from the Wellcome Trust/DBT India Alliance (IA/I/11/2500278), by a grant from DBT (BT/PR2372/AGR/36/696/2011), and intramural grants (IRB project 55, 88, 92, 107, 108, 116).\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe thank Dr. Sudeep Gupta for critically reading the manuscript.\n\n\nSupplementary materials\n\nSequences in italics indicate extra 15 nucleotide tail sequences (junction region). Sequences in bold denotes complementary region between reverse primer of one exon with forward primers of successive exon. 5′ and 3′ represents forward and reverse primer respectively.\n\nReverse complements of the forward sequencing reads of the 915 bp CRE product using genomic DNA extracted from NCI-H1975 cells are displayed as generated by Mutation Surveyor. Panel A: The arrow indicates expected location of the wild-type and T790M mutant allele peak. Panel B: The arrow indicates expected location of the wild-type and L858R mutant allele peak.\n\nReverse complements of the forward sequencing reads of the 915 bp CRE product using genomic DNA extracted from FFPE primary tumor are displayed are displayed as generated by Mutation Surveyor. The arrow indicates expected location of the wild-type and L858R mutant allele peak.\n\n\nReferences\n\nAn X, Lu J, Huang JD, et al.: Rapid assembly of multiple-exon cDNA directly from genomic DNA. PLoS One. 2007; 2(11): e1179. 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PubMed Abstract \| Publisher Full Text\n\nIhle NT, Byers LA, Kim ES, et al.: Effect of KRAS oncogene substitutions on protein behavior: implications for signaling and clinical outcome. J Natl Cancer Inst. 2012; 104(3): 228–39. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKosaka T, Yatabe Y, Endoh H, et al.: Mutations of the epidermal growth factor receptor gene in lung cancer: biological and clinical implications. Cancer Res. 2004; 64(24): 8919–23. PubMed Abstract \| Publisher Full Text\n\nLi S, Li L, Zhu Y, et al.: Coexistence of EGFR with KRAS, or BRAF, or PIK3CA somatic mutations in lung cancer: a comprehensive mutation profiling from 5125 Chinese cohorts. Br J Cancer. 2014; 110(11): 2812–20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLièvre A, Bachet JB, Le Corre D, et al.: KRAS mutation status is predictive of response to cetuximab therapy in colorectal cancer. Cancer Res. 2006; 66(8): 3992–5. PubMed Abstract \| Publisher Full Text\n\nLohinai Z, Hoda MA, Fabian K, et al.: Distinct Epidemiology and Clinical Consequence of Classic Versus Rare EGFR Mutations in Lung Adenocarcinoma. J Thorac Oncol. 2015; 10(5): 738–46. PubMed Abstract \| Publisher Full Text\n\nLynch TJ, Bell DW, Sordella R, et al.: Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med. 2004; 350(21): 2129–39. PubMed Abstract \| Publisher Full Text\n\nMao C, Qiu LX, Liao RY, et al.: KRAS mutations and resistance to EGFR-TKIs treatment in patients with non-small cell lung cancer: a meta-analysis of 22 studies. Lung Cancer. 2010; 69(3): 272–8. PubMed Abstract \| Publisher Full Text\n\nMinton JA, Flanagan SE, Ellard S: Mutation surveyor: software for DNA sequence analysis. Methods Mol Biol. 2011; 688: 143–53. PubMed Abstract \| Publisher Full Text\n\nMitani Y, Nakayama T, Harbers M, et al.: Aptamer-dependent full-length cDNA synthesis by overlap extension PCR. Biotechniques. 2004; 37(1): 124, 126, 128–9. PubMed Abstract\n\nMok TS, Wu YL, Thongprasert S, et al.: Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. N Engl J Med. 2009; 361(10): 947–57. PubMed Abstract \| Publisher Full Text\n\nPaez JG, Jänne PA, Lee JC, et al.: EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science. 2004; 304(5676): 1497–500. PubMed Abstract \| Publisher Full Text\n\nPao W, Miller V, Zakowski M, et al.: EGF receptor gene mutations are common in lung cancers from \"never smokers\" and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad Sci U S A. 2004; 101(36): 13306–11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPao W, Miller VA, Politi KA, et al.: Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med. 2005a; 2(3): e73. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPao W, Wang TY, Riely GJ, et al.: KRAS mutations and primary resistance of lung adenocarcinomas to gefitinib or erlotinib. PLoS Med. 2005b; 2(1): e17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRamteke MP, Patel KJ, Godbole M, et al.: Dataset 1 in CRE: a cost-effective and rapid approach for PCR-mediated concatenation of KRAS and EGFR exons. F1000Research. 2015a. Data Source\n\nRamteke MP, Patel KJ, Godbole M, et al.: Dataset 2 in CRE: a cost-effective and rapid approach for PCR-mediated concatenation of KRAS and EGFR exons. F1000Research. 2015b. Data Source\n\nRamteke MP, Patel KJ, Godbole M, et al.: Dataset 3 in CRE: a cost-effective and rapid approach for PCR-mediated concatenation of KRAS and EGFR exons. F1000Research. 2015c. Data Source\n\nRamteke MP, Patel KJ, Godbole M, et al.: Dataset 4 in CRE: a cost-effective and rapid approach for PCR-mediated concatenation of KRAS and EGFR exons. F1000Research. 2015d. Data Source\n\nSantis G, Angell R, Nickless G, et al.: Screening for EGFR and KRAS mutations in endobronchial ultrasound derived transbronchial needle aspirates in non-small cell lung cancer using COLD-PCR. PLoS One. 2011; 6(9): e25191. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSharma SV, Bell DW, Settleman J, et al.: Epidermal growth factor receptor mutations in lung cancer. Nat Rev Cancer. 2007; 7(3): 169–81. PubMed Abstract \| Publisher Full Text\n\nTuohy TM, Groden J: Exons - introns = lexons: in-frame concatenation of exons by PCR. Hum Mutat. 1998; 12(2): 122–7. PubMed Abstract \| Publisher Full Text\n\nVan Eijk R, Licht J, Schrumpf M, et al.: Rapid KRAS, EGFR, BRAF and PIK3CA mutation analysis of fine needle aspirates from non-small-cell lung cancer using allele-specific qPCR. PLoS One. 2011; 6(3): e17791. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYang JC, Shih JY, Su WC, et al.: Afatinib for patients with lung adenocarcinoma and epidermal growth factor receptor mutations (LUX-Lung 2): a phase 2 trial. Lancet Oncol. 2012; 13(5): 539–48. PubMed Abstract \| Publisher Full Text\n\nZinsky R, Bölükbas S, Bartsch H, et al.: Analysis of KRAS Mutations of Exon 2 Codons 12 and 13 by SNaPshot Analysis in Comparison to Common DNA Sequencing. Gastroenterol Res Pract. 2010; 2010: 789363. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "9749", "date": "31 Jul 2015", "name": "Chandan Kumar", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well conducted proof of principle report that uses the approach of concatenated PCR of exons from genomic DNA to combine exons of two different, clinically relevant genes KRAS and EGFR, to address a clinically relevant question in a resource limiting setting. The assay design and data are provided in sufficient details that any researchers may be able to attempt to carry out similar analyses. There are some comments and suggestions to improve the quality of the manuscript or follow up analyses: Introduction, “These studies have direct implications for carrying out routine KRAS molecular testing along with EGFR mutations for precluding a patient with NSCLC from therapy with EGFR inhibitors, as approved for colorectal cancer (Lievre et al., 2006).”Can the authors cite any reference wherein NSCLC patients are precluded from EGFR inhibitor therapy if they harbor KRAS mutations. The reference cited here is specific to colorectal cancer. Introduction, “Of these direct sequencing is the most commonly used method worldwide”.Is this a personal opinion or there is a reference to support this. Should be cited. Introduction, “concatenation or assembly of individually amplified exons from genomic DNA to generate a cDNA fragment has been described in earlier research”.cDNA specifically refers to complementary DNA derived from RNA through reverse transcriptase. Genomic PCR cannot be said to be used to generate cDNA fragment. I suspect this erroneous phrasing is picked up from a previous reference, but its probably good to not perpetuate the error. Introduction, “Here, we describe a novel methodology to co-amplify all four EGFR exons 18–21 along with KRAS exon 2 in a single multiplex PCR”.It's more like a novel application of a well described methodology supported by several previous references. The novelty, albeit rather incremental, is in combining exons from two different genes, using previously described approach. Should be stated as such. The big appeal of the study is that it affords use of a minimal amount of FFPE sample. Please specify the amount of FFPE material used and yield of DNA to convey an idea of how little/much sample is needed to carry out this analysis. The important consideration of sensitivity has not been addressed. This could be easily tested by assaying a serial dilution of known mutated cell line/FFPE DNA spiked in a wild-type background sample. This will add value to the study. Addition of KRAS codon 61 should be considered as well. Or the difficulty in scaling up should be discussed. How difficult is it to add additional exons. The concatenated PCR product may be amenable to Pyrosequencing to improve sensitivity of detection (particularly in case of low tumor content, low clonality of mutations as is expected in case of dynamically evolving tumors). This should be attempted/ discussed. A direct comparison with the standard technique(s) currently used to test these mutations- in terms of amount of starting material needed, sensitivity of detection, time, and cost will help the argument of the new approach as a superior option.", "responses": [ { "c_id": "1486", "date": "08 Mar 2016", "name": "Amit Dutt", "role": "Author Response", "response": "We thank the reviewer for describing the study as “a well conducted proof of principle report”, and for sharing elaborate comments and suggestions that has significantly improved the quality of the manuscript. Our response to specific concerns are as follows:Referee’s comments: Introduction, “These studies have direct implications for carrying out routine KRAS molecular testing along with EGFR mutations for precluding a patient with NSCLC from therapy with EGFR inhibitors, as approved for colorectal cancer (Lièvre et al., 2006).” Can the authors cite any reference wherein NSCLC patients are precluded from EGFR inhibitor therapy if they harbor KRAS mutations. The reference cited here is specific to colorectal cancer.Author’s response: EGFR and KRAS mutations occur mutually exclusive in NSCLC, which suggests functional redundancy. However, they predict contrasting response rate to tyrosine-kinase inhibitors (TKI). While EGFR mutation predicts longer progression-free survival rate, adverse prognosis is associated with patients harboring KRAS mutations. Thus, the recently reported co-occurrence of KRAS and EGFR activating mutations in 30 of 5125 patients, along with our study of co-occurrence of KRAS and EGFR activating mutations in 3 of 86 patients, raises a clinical concern about the relative value of EGFR and KRAS mutation status as predictors of outcome in NSCLC. As the reviewer may agree these studies may have obvious implications for routine KRAS testing in this disease, potentially precluding EGFR TKI therapy from some patients, similar to current practice in colorectal cancer.In principle, we fully agree with the reviewer that no direct evidence exist to preclude EGFR inhibitor therapy among patients co-harboring EGFR and KRAS mutation. In accordance with the reviewer’s suggestion we have revised the text to reflect the speculative implication of our methodology in NSCLC. Our modified text reads as follows:“….While no evidence exists as yet, these studies may have implications for carrying out routine KRAS molecular testing along with EGFR mutations for precluding a patient with NSCLC from therapy with EGFR inhibitors, as approved for colorectal cancer (Lièvre et al., 2006)….” Referee’s comments: Introduction, “Of these direct sequencing is the most commonly used method worldwide”. Is this a personal opinion or there is a reference to support this. Should be cited.Author’s response: We thank the reviewer for pointing the omission. A relevant citation has been added. However, as our citation in manuscript is likely to be incomplete to summarize the field, some additional studies are mentioned below:Yatabe, Yasushi, et al. \"EGFR Mutation Testing Practices within the Asia Pacific Region: Results of a Multicenter Diagnostic Survey.\" Journal of Thoracic Oncology 10.3 (2015): 438;Angulo, Bárbara, et al. \"A comparison of EGFR mutation testing methods in lung carcinoma: direct sequencing, real-time PCR and immunohistochemistry.\" PLoS One 7.8 (2012): e43842;Ellison, Gillian, et al. \"EGFR mutation testing in lung cancer: a review of available methods and their use for analysis of tumour tissue and cytology samples.\" Journal of clinical pathology 66.2 (2013): 79-89;Cappuzzo, Federico. \"Methods for EGFR Mutation Testing.\" Guide to Targeted Therapies: EGFR mutations in NSCLC. Springer International Publishing, 2014. 19-24. Referee’s comments: Introduction, “concatenation or assembly of individually amplified exons from genomic DNA to generate a cDNA fragment has been described in earlier research”. cDNA specifically refers to complementary DNA derived from RNA through reverse transcriptase. Genomic PCR cannot be said to be used to generate cDNA fragment. I suspect this erroneous phrasing is picked up from a previous reference, but its probably good to not perpetuate the error.Author’s response: We agree and thank the reviewer for bringing it our attention. Our modified text reads as follows:“….concatenation or assembly of individually amplified exons from genomic DNA to generate a coding fragment has been described in earlier research…”. Referee’s comments: Introduction, “Here, we describe a novel methodology to co-amplify all four EGFR exons 18–21 along with KRAS exon 2 in a single multiplex PCR”. It's more like a novel application of a well described methodology supported by several previous references. The novelty, albeit rather incremental, is in combining exons from two different genes, using previously described approach. Should be stated as such.Author’s response: As suggested by the reviewer, we have dropped the term “novel”. Our modified text reads as follows:“….Here, we describe a methodology to co-amplify all four EGFR exons 18–21 along with KRAS exon 2 in a single multiplex PCR….” Referee’s comments: The big appeal of the study is that it affords use of a minimal amount of FFPE sample. Please specify the amount of FFPE material used and yield of DNA to convey an idea of how little/much sample is needed to carry out this analysis.Author’s response: As mentioned in the methodology section subtitled, “Multiplex PCR of KRAS exon 2 and EGFR exons 18-21”, multiplex PCR (50µl per reaction) was carried out in a single tube by using multiplex PCR kit (Qiagen) containing either 10 ng of genomic DNA from the NSCLC cell line or fresh frozen primary tumor, or 50 ng of genomic DNA from FFPE blocks. Furthermore, as mentioned under the methodology section subtitled, “Concatenation of exons and sequencing analysis”, 2 µl of multiplex PCR product was used as template in a 50 µl PCR reaction for concatenation. Referee’s comments: The important consideration of sensitivity has not been addressed. This could be easily tested by assaying a serial dilution of known mutated cell line/FFPE DNA spiked in a wild-type background sample. This will add value to the study.Author’s response: As mentioned under the last paragraph of the discussion section, “….As the limitation of the CRE strategy is defined by the sensitivity and resolution of the sequencing methodology adopted…” – which in this study has been Sanger Sequencing-- as the sensitivity of Sanger Sequencing is well established from FFPE and mutated cell line, we humbly differ from the reviewer that admixture experiment would add additional information. Referee’s comments: Addition of KRAS codon 61 should be considered as well. Or the difficulty in scaling up should be discussed. How difficult is it to add additional exons.Author’s response: KRAS codon 12 is mutated at a frequency of 25-50% in Caucasian population and 5-15% among East Asians. In a recent study we reported 18.6% KRAS codon 12 among Indian population (n=86). Given that KRAS codon 61 mutation exist at frequency < 1%; and, that none were found in our study in 86 patients, we decided to not include KRAS codon 61 mutation in this study to only present the proof of principle of the CRE methodology. However, we do agree with the reviewer about the significance of KRAS codon 61 mutation, and do hope to include it along with other known activating mutations in NSCLC.We submit that based on literature, additional exons can be added to the current methodology, as at least up to 10 genomic spliced exons fragment of 2295 bp has been described in literature using similar methodology. Referee’s comments: The concatenated PCR product may be amenable to Pyrosequencing to improve sensitivity of detection (particularly in case of low tumor content, low clonality of mutations as is expected in case of dynamically evolving tumors). This should be attempted/ discussed.Author’s response: We fully agree with the reviewer’s insights that CRE can be utilized at high throughput mode to determine complete spectrum of EGFR and KRAS mutations using targeted next generation sequencing. Consistent with the reviewer’s suggestion the last paragraph of the discussion section reads, “… the limitation of the CRE strategy is defined by the sensitivity and resolution of the sequencing methodology adopted, concatenated EGFR and KRAS PCR products from multiple individuals—each tagged with unique bar code sequence—can be pooled and deep-sequenced using a NGS platform. The CRE strategy described here can reduce the labor and cost of performing individual PCR for all exons for each patient and effectively circumvent the noise due to variation in normalization for equimolar pooling of exons within the same sample at a resolution of single base.” Referee’s comments: A direct comparison with the standard technique(s) currently used to test these mutations- in terms of amount of starting material needed, sensitivity of detection, time, and cost will help the argument of the new approach as a superior option. Author’s response: As detailed in the manuscript, this proof of principle study introduces CRE as a methodology involving single multiplex-PCR followed by concatenation of the PCR product as one linear fragment for direct sequencing, as opposed to 5 rounds of PCR reaction followed by 10 rounds of sequencing reactions. A systematic comparative analysis is currently underway at our center using clinical cancer specimens for CRE compared to Sanger sequencing based methodology; SNaPShot PCR; Cobas system; Mass spec genotyping on a larger cohort sample, beyond the scope of this manuscript. Hence, we express our inability to include analysis from this ongoing study at this early on stage.We sincerely thank the reviewer for the elaborate and detailed constructive review. Hope the reviewer will find the improved version of the manuscript acceptable for indexation." } ] }, { "id": "9740", "date": "24 Feb 2016", "name": "Bob T. Li", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the age of precision medicine with an expanding number of oncogenic drivers in lung cancers that may be treated with targeted agents, multiplexed genomic testing is increasingly important in clinical practice. The study by Ramteke et al. describes a rapid and relatively inexpensive multiplexed test for EGFR and KRAS mutations. The methods are well described and the test is of clinical relevance, particularly in settings with limited resources and without access to tumor next generation sequencing. I recommend making the following minor revisions:In the introduction, it is incorrect to suggest that the reason for KRAS testing in lung cancers is to preclude patients from EGFR inhibitors. The rationale for EGFR inhibitors in lung cancers is very different to that of colorectal cancers, as activating EGFR mutations in lung cancers predict response to EGFR TKIs. However, it is still important to test all lung cancers for KRAS mutations as it is a common oncogenic driver occuring in over 25% of lung adenocarcinomas. Being a driver KRAS is highly unlikely to co-exist with other actionable drivers, therefore once KRAS is found one could justify that further genomic testing for other drivers is not necessary, especially in a resource limited setting. It should be acknowledged that the authors' CRE method will not capture all KRAS mutations, especially KRAS mutations in exon 3 codon 61. However, the ability to capture the majority of KRAS and EGFR mutations in one single inexpensive test is still of value for patients with lung cancers.", "responses": [ { "c_id": "1833", "date": "08 Mar 2016", "name": "Amit Dutt", "role": "Author Response", "response": "We sincerely thank the reviewer for the elaborate and detailed constructive review. In particular, we are grateful to the reviewer for describing the study as “a well conducted proof of principle report”. We hope the reviewer will find the improved version of the manuscript acceptable, without reservation. Our response to specific concerns are as follows-Referee’s comment 1: Introduction, “These studies have direct implications for carrying out routine KRAS molecular testing along with EGFR mutations for precluding a patient with NSCLC from therapy with EGFR inhibitors, as approved for colorectal cancer (Lievre et al., 2006).” Can the authors cite any reference wherein NSCLC patients are precluded from EGFR inhibitor therapy if they harbor KRAS mutations. The reference cited here is specific to colorectal cancer. Author’s response: EGFR and KRAS mutations occur mutually exclusive in NSCLC, which suggests functional redundancy, however they predict contrasting response rates to tyrosine-kinase inhibitors (TKI). While EGFR mutation predicts longer progression-free survival rate, adverse prognosis is associated with patients harboring KRAS mutations. Thus, the recently reported co-occurrence of KRAS and EGFR activating mutations in 30 of 5125 patients, along with our study of co-occurrence of KRAS and EGFR activating mutations in 3 of 86 patients, raises questions about the relative value of EGFR and KRAS mutation status as predictors of outcome in NSCLC. As the reviewer may agree these studies may have obvious implications for routine KRAS testing in this disease, potentially precluding EGFR TKI therapy from some patients, similar to current practice in colorectal cancer. However, their direct mention in NSCLC is speculative. Thus, in principle, we fully agree with the reviewer that no direct evidence exists to preclude EGFR inhibitor therapy among patients co-harboring EGFR and KRAS mutation. In accordance with the reviewer’s suggestion we have revised the text to reflect the speculative implication of our methodology in NSCLC. Our modified text reads as follows:“….While no evidence exists as yet, these studies may have implications for carrying out routine KRAS molecular testing along with EGFR mutations for precluding a patient with NSCLC from therapy with EGFR inhibitors, as approved for colorectal cancer (Lievre et al., 2006)….” Referee’s comment 2: Introduction, “Of these direct sequencing is the most commonly used method worldwide”. Is this a personal opinion or there is a reference to support this. Should be cited. Author’s response: We thank the reviewer for pointing the omission. A relevant citation has been added. However, as our citation in manuscript is likely to be incomplete to summarize the field, some additional studies are mentioned below: Yatabe, Yasushi, et al. \"EGFR Mutation Testing Practices within the Asia Pacific Region: Results of a Multicenter Diagnostic Survey.\" Journal of Thoracic Oncology 10.3 (2015): 438; Angulo, Bárbara, et al. \"A comparison of EGFR mutation testing methods in lung carcinoma: direct sequencing, real-time PCR and immunohistochemistry.\" PLoS One 7.8 (2012): e43842; Ellison, Gillian, et al. \"EGFR mutation testing in lung cancer: a review of available methods and their use for analysis of tumour tissue and cytology samples.\" Journal of clinical pathology 66.2 (2013): 79-89; Cappuzzo, Federico. \"Methods for EGFR Mutation Testing.\" Guide to Targeted Therapies: EGFR mutations in NSCLC. Springer International Publishing, 2014. 19-24. Referee’s comment 3: Introduction, “concatenation or assembly of individually amplified exons from genomic DNA to generate a cDNA fragment has been described in earlier research”. cDNA specifically refers to complementary DNA derived from RNA through reverse transcriptase. Genomic PCR cannot be said to be used to generate cDNA fragment. I suspect this erroneous phrasing is picked up from a previous reference, but its probably good to not perpetuate the error. Author’s response: We agree and thank the reviewer for bringing it our attention. Our modified text reads as follows:“….concatenation or assembly of individually amplified exons from genomic DNA to generate a coding fragment has been described in earlier research…”. Referee’s comment 4: Introduction, “Here, we describe a novel methodology to co-amplify all four EGFR exons 18–21 along with KRAS exon 2 in a single multiplex PCR”. It's more like a novel application of a well described methodology supported by several previous references. The novelty, albeit rather incremental, is in combining exons from two different genes, using previously described approach. Should be stated as such. Author’s response: As suggested by the reviewer, we have dropped the term “novel”. Our modified text reads as follows:“….Here, we describe a methodology to co-amplify all four EGFR exons 18–21 along with KRAS exon 2 in a single multiplex PCR….” Referee’s comment 5: The big appeal of the study is that it affords use of a minimal amount of FFPE sample. Please specify the amount of FFPE material used and yield of DNA to convey an idea of how little/much sample is needed to carry out this analysis. Author’s response: As mentioned in the methodology section subtitled, “Multiplex PCR of KRAS exon 2 and EGFR exons 18-21”, multiplex PCR (50µl per reaction) was carried out in a single tube by using multiplex PCR kit (Qiagen) containing either 10 ng of genomic DNA from the NSCLC cell line or fresh frozen primary tumor, or 50 ng of genomic DNA from FFPE blocks. Furthermore, as mentioned under the methodology section subtitled, “Concatenation of exons and sequencing analysis”, 2 µl of multiplex PCR product was used as template in a 50 µl PCR reaction for concatenation. Referee’s comment 6: The important consideration of sensitivity has not been addressed. This could be easily tested by assaying a serial dilution of known mutated cell line/FFPE DNA spiked in a wild-type background sample. This will add value to the study. Author’s response: As mentioned under the last paragraph of the discussion section, “….As the limitation of the CRE strategy is defined by the sensitivity and resolution of the sequencing methodology adopted…” – which in this study has been Sanger Sequencing, but could significantly vary if advanced contemporary sequencing methodologies are adopted. However, as the sensitivity of PCR followed by directed Sanger Sequencing is well established from FFPE samples and mutated cell line, we humbly differ from the reviewer that admixture experiment would add additional information. Referee’s comment 7: Addition of KRAS codon 61 should be considered as well. Or the difficulty in scaling up should be discussed. How difficult is it to add additional exons. Author’s response: KRAS codon 12 is mutated at a frequency of 25-50% in Caucasian population and 5-15% among East Asians. In a recent study we reported 18.6% KRAS codon 12 among Indian population (n=86)-- (Choughule et al., 2014). Given that KRAS codon 61 mutation exist at frequency < 1%; and, that none were found in our study in 86 patients, we decided to not include KRAS codon 61 mutation in this study to only present the proof of principle of the CRE methodology. However, we do agree with the reviewer about the significance of KRAS codon 61 mutation, and do hope to include it along with other known activating mutations in NSCLC in an improved version of CRE.However, to reflect the pertinent suggestion made by the reviewer we have modified our discussion to read as follows: “…Additionally, the current version of CRE is limited by exclusion of fewer number of exons of EGFR and KRAS. Inclusion of known extracellular EGFR and KRAS exon 3 codon 61 mutation may help to immediately expand the scope of its application to other cancers, such as glioblastoma.” Referee’s comment 8: The concatenated PCR product may be amenable to Pyrosequencing to improve sensitivity of detection (particularly in case of low tumor content, low clonality of mutations as is expected in case of dynamically evolving tumors). This should be attempted/ discussed. Author’s response: We fully agree with the reviewer’s insights that CRE can be utilized at high throughput mode to determine complete spectrum of EGFR and KRAS mutations using targeted next generation sequencing. Consistent with the reviewer’s suggestion the last paragraph of the discussion section, “… the limitation of the CRE strategy is defined by the sensitivity and resolution of the sequencing methodology adopted, concatenated EGFR and KRAS PCR products from multiple individuals—each tagged with unique bar code sequence—can be pooled and deep-sequenced using a NGS platform. The CRE strategy described here can reduce the labor and cost of performing individual PCR for all exons for each patient and effectively circumvent the noise due to variation in normalization for equimolar pooling of exons within the same sample at a resolution of single base.” Referee’s comment 9: A direct comparison with the standard technique(s) currently used to test these mutations- in terms of amount of starting material needed, sensitivity of detection, time, and cost will help the argument of the new approach as a superior option. Author’s response: As detailed in the manuscript, this proof of principle study introduces CRE as a methodology involving single multiplex-PCR followed by concatenation of the PCR product as one linear fragment for direct sequencing, as opposed to 5 rounds of PCR reaction followed by 10 rounds of sequencing reactions. A systematic comparative analysis is currently underway at our center using clinical cancer specimens for CRE compared to Sanger sequencing based methodology; SNaPShot PCR; Cobas system; Mass spec genotyping on a larger cohort sample, beyond the scope of this manuscript. Hence, we express our inability to include analysis from this ongoing study at this early on stage." }, { "c_id": "1834", "date": "08 Mar 2016", "name": "Amit Dutt", "role": "Author Response", "response": "We sincerely thank reviewer for approving our submission. We are particularly grateful to the reviewer for describing the study as, “The methods are well described and the test is of clinical relevance, particularly in settings with limited resources and without access to tumor next generation sequencing”. The suggestions made by the reviewers have contributed to an improved version of the manuscript. Specifically, we have, in the revised version:Referee’s comment 1: In the introduction, it is incorrect to suggest that the reason for KRAS testing in lung cancers is to preclude patients from EGFR inhibitors. The rationale for EGFR inhibitors in lung cancers is very different to that of colorectal cancers, as activating EGFR mutations in lung cancers predict response to EGFR TKIs. However, it is still important to test all lung cancers for KRAS mutations as it is a common oncogenic driver occuring in over 25% of lung adenocarcinomas. Being a driver KRAS is highly unlikely to co-exist with other actionable drivers, therefore once KRAS is found one could justify that further genomic testing for other drivers is not necessary, especially in a resource limited setting. Author’s response: As described in or response to Reviewer 1’s first comment, we agree we with the reviewer that no direct evidence exists to preclude EGFR inhibitor therapy among patients co-harboring EGFR and KRAS mutation. In accordance with the reviewer’s suggestion we have revised the text to reflect the speculative implication of our methodology in NSCLC. Our modified text reads as follows:“….While no evidence exists as yet, these studies may have implications for carrying out routine KRAS molecular testing along with EGFR mutations for precluding a patient with NSCLC from therapy with EGFR inhibitors, as approved for colorectal cancer (Lievre et al., 2006)….” Referee’s comment 2: It should be acknowledged that the authors' CRE method will not capture all KRAS mutations, especially KRAS mutations in exon 3 codon 61. However, the ability to capture the majority of KRAS and EGFR mutations in one single inexpensive test is still of value for patients with lung cancers.Author’s response: As described in our response to Reviewer 1’s comment 7, we agree with the reviewer about the significance of KRAS codon 61 mutation, and do hope to include it along with other known activating mutations in NSCLC. However, to reflect the pertinent suggestion made by the reviewer we have modified our discussion to read as follows: “…Additionally, the current version of CRE is limited by exclusion of fewer number of exons of EGFR and KRAS. Inclusion of known extracellular EGFR and KRAS exon 3 codon 61 mutation may help to immediately expand the scope of its application to other cancers, such as glioblastoma.”" } ] } ]	1	https://f1000research.com/articles/4-160
https://f1000research.com/articles/5-294/v1	07 Mar 16	{ "type": "Opinion Article", "title": "Junior biomedical scientists and preprints", "authors": [ "Gary McDowell" ], "abstract": "Researchers, publishers and representatives of funding agencies gathered at ASAPBio (February, 2016) to discuss the use of preprint publications in biology. It became clear through the discussion on Twitter with #ASAPBio that many were unclear as to the purpose of the meeting, how preprints could help or hinder junior scientists, or even what preprints are. As a postdoc attendee of the meeting, I would like to share some information and resources to help junior scientists decide for themselves whether submitting preprints are a worthwhile endeavor, and explain why I, and the lab I am currently part of, have decided to start publishing our papers on preprint servers when also submitting to traditional journals.", "keywords": [ "preprint", "publishing", "peer review", "journals", "biomedicine" ], "content": "What’s a preprint?\n\nA preprint is a manuscript in a finished form that has not yet been published in a traditional journal. It is essentially the document that you submit to a traditional journal for peer review, deposited on a server for anyone to access. The practice of publishing preprints is universal in the fields of physics and mathematics since the creation of the ArXiv preprint server in 1991, but the biological sciences have been slower to take up the preprint archiving model1,2. A biological sciences preprint server, bioRxiv, is already in existence, and well used. At ASAPBio, Paul Ginsparg, founder of ArXiv, gave a keynote talk about the history of ArXiv which you can watch on the video stream. iBiology has also released a video with Youreka Science which explains what preprints are. Preprint publication, it has been proposed, could accelerate the dissemination of work and the pace of discovery3 (also in preprint format!).\n\n\nWhat is being asked of the community?\n\nThere were many discussions on social media surrounding the ASAPBio meeting and aspects of scientific publishing; so many, in fact, that the original intention of the meeting was in danger of being drowned out in the noise. The focus of this meeting, like this paper, was to convince scientists to submit publications to preprint servers such as bioRxiv, before or at the same time as submission to a traditional journal of choice: adding a small step to the normal publication workflow. The goal of the meeting was to make sure that as many barriers as possible to that process could be removed, at journals, funding agencies and within the culture of biomedicine.\n\n\nWhy will I (as a junior scientist) preprint?\n\nFrom the perspective of a junior scientist, there are several reasons that have convinced me to deposit my publications as preprints in the future (and, incidentally, have also convinced my current lab to do the same).\n\nPreprints are another opportunity, along with talks, seminars, public lectures and blog posts, to disseminate my work. Because preprints are free and open, I can share it on websites, through social media, to researchers in other countries, to friends and family. Being able to share my work for discussion has always been useful both for improving my science and also for letting people hear about my work. The work can be shared immediately, and can be useful for others to consider in the context of their own work, whilst traditional peer review is taking place. It adds to, and does not detract from, my productivity as a scientist.\n\nPreprints provide evidence of productivity on applications for grants, fellowships, and jobs. Throughout various points of training, there are markers for junior scientists where they need publications to pass to the next phase or receive funding, and the time taken to publish is affecting the time junior scientists spend in training positions. For example, I have heard recommendations that postdocs should apply for a K99/R00 mechanism in the second year of their postdoctoral work, which directly contradicts the expectation that a first-author publication from that work is invariably required to achieve a good score. I have heard similar expectations exist in various funding mechanisms for trainees, through to the NSF Graduate Research Fellowship Program that undergraduates and early graduate students are eligible for. Indeed, with publication requirements for graduating from PhD programs, and, increasingly, an expectation that undergraduates will have publications for graduate fellowships or even applying to graduate school, trainees are taking longer in training positions, or taking time out after undergraduate degrees to work as technicians, simply waiting for publications to be accepted. Preprints give a marker of productivity and moving towards publication that could relieve pressure at this point and subsequently reduce unnecessary time in training. What was most encouraging at ASAPBio (especially as I had the good fortune be in the subgroup on the role of funding agencies with representatives from EMBO, HHMI, NIH, NSF and the Wellcome Trust) was that funding agencies were on board with recognizing a preprint as a product. Representatives from EMBO suggested that they already informally uses these kinds of markers for renewing postdoctoral fellowship applications, because they do not think it is reasonable to expect postdocs to have published by the time of renewal. The NIH Biosketch potentially already allows for these kinds of products to be included as evidence4, although clarification on this is needed and the NIH is due to issue a statement in March. Moving forward the hope is that these products will be more commonly recognized as a marker of productivity and progression, rather than relying on the stop-and-start flow of accepted publications.\n\nI will lay out my bias here, however. I try to make the conscious decision, when it is mine to make, not to join the impact factor dance in publishing (when I have done so, it has been at points when it has been worth a try for applications, or with collaborators who feel similarly pressured to do so). To me, it makes sense to have a publicly available work that I can solicit feedback on, and then also submit the paper to a journal which I think fits and more importantly, which I think will accept it without much hassle, but which I think will reach the audience for which it is intended. I do this not only because of personal preference (I certainly realize it is an idealistic way of publishing) but because there are many, many postdocs with excellent publications, many in high impact factor journals, competing desperately for jobs and I truly don’t see it being worth the effort, and just want to publish consistently and regularly. The metric of the impact factor has become inflated; and the devolution of decision making on what makes good science to the realm of a few journals and the sheer number of applicants for positions could be resulting in fewer papers actually being read, because reviewers simply can’t read everything from everyone (for commentary and thoughts on this please see relevant discussions on the subject e.g. 5–7). A preprint can give the most recent snapshot of your work and productivity (as your most recent publication could easily be work that’s 2 years old) and could be a useful product in actually evaluating someone’s science directly as part of an application, if reviewers have time to read only a selection of your work. Arguably, a work that has not been through the peer review process and is recent can give a much more realistic snapshot of you as a scientist.\n\n\nWhat are the reasons not to preprint?\n\nPhilosophical reasons given against preprinting generally revolve around: questions of whether they are recognized products or markers of productivity; the possibility of being scooped; and settling the issue of who has priority over a novel discovery.\n\nTo these, my simple answer was to talk to a physicist. I happened to see a tenured colleague from the physics department at Tufts on my return and explained what ASAPBio was about. When they asked why biologists don’t use preprints, and I gave the answers above, they replied that those were precisely the reasons they use preprints. In grants and tenure review, people recognize that the publication process is not under your control, but producing finished manuscripts and depositing them on a server, where they can be read, and then letting the publication process take its course is taken as an obvious part of the continuing process of science. In terms of being scooped, the manuscript has a DOI and is publicly available, and my physicist colleague told me they use preprints to explicitly avoid being scooped. In terms of priority of discovery, I have to agree with the view that publishing preprints by itself isn’t going to resolve or cause such disputes, unless the field decides to ignore contextual evidence for work in a scientific discovery. I must declare that establishing priority of discovery is not a personal goal, or certainly not one I am particularly concerned about in science, and so am perhaps not the best person to answer to that point, but there is a commentary piece at ASAPBio that directly addresses this topic.\n\nIn more practical terms, not all journals will accept preprints, but the vast majority do, with the exception of Cell Press, who publicly stated that preprints were possible to submit only through a vague, non-transparent process of contacting the editor to discuss the matter, with the caveat that the preprint should not have “been noticed” by the field. That they took this opportunity in front of an audience of junior scientists in particular was very surprising, especially as many of the other high impact factor journals, such as Science, Nature, PNAS, PLoS and others as described here in this list on Wikipedia will accept preprints. Indeed a group of publishers have drafted a statement actively encouraging the use of preprints and one of the commentary pieces for ASAPBio came from Genetics and G3 on the synergy that preprints and traditional publications could create. I will state here again for clarification that although there was an active discussion about post-publication peer review and extending that to the end of journals altogether, that was only one of the many areas of discussion and not the goal of the meeting, despite what might have been the impression.\n\nThere was also some discussion about health sciences, and publishing data that may give false hope to patients or be damaging if made publically available prior to peer review. On this point I will simply refer to Stephen Curry’s piece in the Guardian on the Zika virus, and the apparent embrace of open research in that context. We could well ask, why is this not the case for all research, not just for Zika, and recently, Ebola? The consensus at the meeting seemed to be that it was more, not less, important for medical research to be disseminated openly and rapidly. There have however been recent conflicts over the concept of “research parasitism” and data sharing in the medical literature8 and the battle for open data in the health sciences is a problem in itself. However I would argue that if you have submitted a piece for publication in a journal, then you are confident of those results and there shouldn’t be any serious harm (or any errors may be spotted more quickly, potentially) in a preprint of the article.\n\nThis comes down to a lot of what people seem to fear: which is that the literature will be overwhelmed with junk. David Stern wrote a commentary directly addressing this; essentially, it is very unlikely that people who publish junk will be rewarded for doing so. The peer review process is not perfect – no peer review process, to my eyes, is perfect – but added opportunities for review, dissemination, and replication can surely only benefit the scientific process. In addition, as Stern points out, your name is your brand and if you publish junk, your brand will be associated with junk. I would never (consciously) publish junk just to have a piece available and especially not in such an incredibly open and public format. The risks associated with doing so would be too great compared to depositing work I would stand behind publicly.\n\n\nWhat next?\n\nI have told you what I am doing and given my opinion and position after attending the meeting; but this is only to encourage more discussion. I and others, such as Samuel L. Díaz-Muñoz who wrote this commentary on a practical guide for young scientists, and James Fraser who wrote a commentary from the perspective of a junior faculty member, are interested in finding out what our community needs or wants, and hopes or fears about preprints. My impression is that firstly most people have not heard about preprints; my second is that there are some misconceptions about what they (and this meeting) were about. I hope this piece has given some clarification on these points but I am aware of my romantic disposition towards doing my science and am also in an environment and field where I don’t feel that some of the pressures people are most concerned about apply.\n\nI am also not a fan of the current push towards “people not projects” as a model that should define biomedical research. Whilst there can be a place for such models, to help those who thrive in that kind of environment, we are pushing ever further into evaluating science on metrics and backgrounds of people, and looking less and less at the science itself. Preprints can provide evidence of the work and mind of the scientist in a more up-to-date fashion and, divorced from the impact factor of a particular journal, the work itself must be considered.\n\nUltimately I am excited about increasing the transparency and availability of science; increasing access to my work and enabling more discussion about its implications and improvement; and adding to the products that I can point to as evidence of my scientific endeavors in an attempt to reduce the time spent simply waiting around for publications to come out before applying for jobs and grants. Science is a continuous and reformative process and to me there is a great value in having science openly available for critique as well as having close scrutiny enforced through various models of peer review.", "appendix": "Competing interests\n\n\n\nNo competing interests were declared.\n\n\nGrant information\n\nThe author declared that no grants were involved in supporting this work.\n\n\nReferences\n\nMarshall E: Varmus defends E-biomed proposal, prepares to push ahead. Science. 1999; 284(5423): 2062–3. PubMed Abstract \| Publisher Full Text\n\nDesjardins-Proulx P, White EP, Adamson JJ, et al.: The case for open preprints in biology. PLoS Biol. 2013; 11(5): e1001563. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVale RD: Accelerating scientific publication in biology. Proc Natl Acad Sci U S A. 2015; 112(44): 13439–46. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRockey S: Changes to the Biosketch. NIH Extramural Nexus, [cited 2016 Feb 26], 2014. Reference Source\n\nWhat do search committees really read in a CV during initial screening for a faculty position? Academia Stack Exchange, [cited 2016 Feb 26]. Reference Source\n\nHow North American ecology faculty position search committees work. Dynamic Ecology, [cited 2016 Feb 26]. Reference Source\n\nOur faculty search so far. Cryptogenomicon, [cited 2016 Feb 26]. Reference Source\n\nLongo DL, Drazen JM: Data Sharing. N Engl J Med. 2016; 374(3): 276–277. PubMed Abstract \| Publisher Full Text" }	[ { "id": "12947", "date": "18 Mar 2016", "name": "Needhi Bhalla", "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 appreciated the author explaining the role that preprints could play in biological and biomedical research and support publication. I have several comments that I think could make this a stronger opinion article.“A preprint is a manuscript in a finished form that has not yet been published in a traditional journal.” I would emphasize in this first paragraph that preprints are not peer-reviewed, particularly since the author points out that preprint posting is likely to be simultaneous or soon after submission to a traditional journal. “The practice of publishing preprints is universal in the fields of physics and mathematics”According to Lariviere, et. al, 2014, only 20% of published papers in the general field of physics are posted as preprints on arXiv. While it is nearly universal in some physics subfields, i.e astronomy, astrophysics and nuclear and particle physics (60-70% of journal articles are posted as preprints), it is less common to post a preprint and subsequently publish in a peer-reviewed journal in fields such as solid state physics. Rather than undermining the value of preprints, this disparity highlights that posting a preprint can be a considered decision rather than a common practice even in the field of physics, something that may also be eventually true in biological and biomedical research. I appreciated the author’s personal perspective on publishing. Another point that may argue against the \"junk\" argument: Several studies have evaluated the impact, in terms of citations, of papers posted on arXiv and subsequently published in journals versus papers only published in journals and found no difference in the level of impact (Davis and Fromerth, 2007; Gentil-Beccot et al., 2009; Lariviere et al., 2014).", "responses": [] }, { "id": "14378", "date": "15 Jun 2016", "name": "Ron Vale", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI think that this article is a useful contribution and would recommend others to read it as well. I should disclose that I am one of the organizers of the ASAPbio meeting, so perhaps that colors my opinion of the article, which describes outcomes of the meeting. However, I do feel that McDowell has done a very good job separating information from his personal opinion. Both are useful to know and his clear wording defines that boundary. McDowell also has done a good job describing the goals of the meeting and the main topics discussed. There was a reasonable amount of confusion on social media, and thus I think that it is valuable to have McDowell clearly spell out the main themes. Furthermore, a complete record of the meeting can be found here, in cases readers want to go directly to the record: http://asapbio.org/meeting-information\n\nThis article is largely written as a meeting report and an opinion piece. But it is particularly timely and important to have the voices of young scientists (like McDowell), especially in essay form. Junior scientists are suffering the most from the current publication system. But they also are the most vulnerable in their careers to managing any changes that might be introduced. They are also the ones who will inherent any new science communication system that will be built. Thus, junior scientists (not just senior scientists like myself) should be given the opportunity to write articles on this matter, such as the thoughtful piece by McDowell. The general message is that preprints are manageable and adaptation to a world of preprints and journals is not hard and indeed very welcome by junior scientists. I need not micromanage the content of this article with my review, especially as the information is accurate and the boundary between opinion and information is well defined. There is a lot of interesting material here in the piece, including McDowell’s discussion of the dilemma of the K99 NIH and publication (and how preprints might help) and the subject of scientific priority. Needhi Bhalla (another referee) also raised points of discussing more about journal submissions in the physics community. Paul Ginsparg discussed this point in his ASAPbio talk and you can download his slides next to his talk on the agenda (or also watch his talk): http://asapbio.org/meeting-information/schedule-location\nBut to quote from his slide 13:” ’07-’14 data (HEP): > 80% with journal ref; vast majority of rest subject to some form of review (conf proceedings, theses, lecture notes)”. Thus the vast majority of work going to arXiv goes to journals and the remainder also are mostly subject to some form of peer review. The same scenario is likely to happen in biology.", "responses": [] } ]	1	https://f1000research.com/articles/5-294
https://f1000research.com/articles/5-292/v1	07 Mar 16	{ "type": "Research Article", "title": "A pilot trial to examine the association between circulating endothelial cell levels and vascular injury in patients with diabetes and chronic kidney disease", "authors": [ "Shayan Shirazian", "Candace Grant", "Vikash Rambhujun", "Ritika Sharma", "Ronak Patel", "Shahidul Islam", "Joseph Mattana", "Candace Grant", "Vikash Rambhujun", "Ritika Sharma", "Ronak Patel", "Shahidul Islam", "Joseph Mattana" ], "abstract": "ObjectiveWhile albuminuria is a marker for progressive chronic kidney disease (CKD) in patients with type 2 diabetes (T2DM), both albuminuric and normoalbuminuric patients appear prone to vascular injury. This pilot study examines the association between circulating endothelial cell (CEC) levels and vascular injury in patients with T2DM and CKD.MethodsIn this cross-sectional study, eligible adult patients had T2DM, and stage 3 CKD (estimated glomerular filtration rate between 30 and 60 mL/min/1.73m2). CEC levels were tested by Janssen Diagnostics, LLC using an immuno-magnetic bead-based assay. CEC levels were compared to levels in a previously tested normal population. Correlations between CEC levels and other vascular injury markers (urine albumin, von-Willebrand factor antigen, hs-CRP, uric acid) were performed.ResultsPatients included 40 adults of which nineteen were normoalbuminuric. Mean CEC levels (38.7, SD 38.1 cells) were significantly higher than the normal population (M = 21±18 cells, p<0.001; N = 249), including in the normoalbuminuric subgroup (M = 42.9±42.5 cells, p<0.001). CEC levels were significantly correlated with uric acid levels (r=0.33, p=0.039).ConclusionsCEC levels in patients with T2DM and CKD, both albuminuric and normoalbuminuric, are significantly higher than a normal population, suggesting the presence of vascular injury in both groups. Future studies are needed to evaluate the role of CECs as a biomarker to predict outcomes in normoalbuminuric patients with CKD.", "keywords": [ "Circulating Endothelial Cells", "Diabetes", "Chronic Kidney Disease", "Vascular Injury" ], "content": "Introduction\n\nType 2 diabetes (T2DM) is the most common cause of pre-dialysis chronic kidney disease (CKD) in the United States affecting approximately 10 million Americans1,2. Of these patients with T2DM and CKD only a small proportion will progress to end stage renal disease (ESRD) requiring dialysis or transplant2. Identifying patients with T2DM and CKD at high risk for progressive renal disease could lead to a decrease in the incidence of ESRD through the implementation of interventions known to slow progression3. Unfortunately, identifying which of these patients are at high risk remains a challenge, due to inaccurate biomarkers of progressive renal disease.\n\nUrine microalbumin levels are the most commonly used biomarker for progressive diabetic renal damage4. Despite this, they have been found to be a poor predictor of progression to ESRD in T2DM, and are in the normal range in up to 50% of patients with T2DM and CKD5–11. Patients with diabetes and normoalbuminuria are at risk for progressive renal function decline12, and those with diabetes, CKD and normoalbuminuria are at higher risk of cardiovascular events and all-cause mortality than diabetes patients without CKD13. Furthermore, patients with T2DM, CKD and normoalbumuria have a similar prevalence of micro and macroangiopathies as patients with T2DM and albuminuria7. Biomarkers that quantify underlying vascular injury and predict future renal and cardiovascular risk independent of urine microalbumin are needed in this population7,14.\n\nCirculating endothelial cells (CECs) are a reliable serologic marker of vascular injury. These cells have been found to detach and circulate in human sera after sustained activation by inflammatory and thrombotic stimuli15. Levels of these cells correlate with the degree of vascular injury, with higher levels seen in patients with atherosclerotic cardiovascular disease, small vessel vasculitis, renal transplant rejection, metastatic carcinoma and advanced chronic kidney disease15–18. In patients with T2DM and CKD, CEC levels may quantify the degree of underlying vascular damage independent of urine microalbumin, potentially acting as a seromarker for future renal or cardiovascular events. The purpose of this study was to determine whether CEC levels correlate with other markers of vascular injury in patients with T2DM and CKD including those with normoalbuminuria.\n\n\nMethods\n\nThis was designed as a cross-sectional study of CEC levels and other vascular injury markers in patients with T2DM and CKD. Recruitment occurred at the outpatient nephrology offices of a large northeastern hospital. This office has an annual caseload of approximately 1500 patients with T2DM and CKD, of whom 25% are minorities (black or hispanic). The study was approved by the local institutional review board (local board reference number 13002, IRBNet ID – 403557), and all patients gave their written informed consent prior to study procedures.\n\nPatients aged 18–89 years with T2DM for ≥ 8 years, and stage 3 CKD with an estimated GFR between 30 and 60 mL/min/1.73m2 were considered for study inclusion. This inclusion criterion selected for a population at high risk for vascular injury that had not yet progressed to advanced CKD. Patients with prior evidence of renal artery stenosis, biopsy proven non-diabetic etiology of kidney disease, recurrent urinary tract infections, microhematuria, active malignancy, recent hospitalization and nephrotic range proteinuria were excluded. The exclusion criterion selected for patients with the highest likelihood of having diabetes related renal vascular injury, minimizing the possibility of elevations in CEC levels caused by alternative etiologies. Patients were selected to ensure approximately equal numbers of males and females, and normoalbuminuric and albuminuric subgroups.\n\nTwelve hundred electronic charts with the ICD 9 coding diagnoses of T2DM and stage 3 CKD were selected for review of inclusion/exclusion criteria. Of these charts, 800 patients met all inclusion/exclusion criteria and 103 consecutive patients were asked to participate in the study when they presented for a follow-up appointment at our outpatients offices. Of those patients who were approached, 40 (38%) signed consent forms and participated in a single study session apart from their regularly scheduled follow-up visit.\n\nAll study procedures were performed during a single, morning 1-hour study session. For all patients, a complete history and physical including seated blood pressure was performed and baseline characteristics were recorded. For blood pressure readings, the lower of two seated measurements taken after 5 minutes of sitting was recorded. Comorbidities were defined by patient report or documentation in the patient’s problem list. Cardiovascular disease was defined as a prior history of coronary artery disease (CAD), congestive heart failure (CHF), peripheral vascular disease (PVD) or stroke.\n\nBlood and urine samples were collected from all patients at the time of recruitment. This included serum creatinine, uric acid, von Willebrand Factor antigen levels, high sensitivity C-reactive protein, morning urine microalbumin to creatinine ratios (UACR) and CEC levels. For low density lipoprotein (LDL) cholesterol, hemoglobin A1c (HbA1c), serum phosphorus and blood hematocrit, the most recent value from outpatient blood work taken within 6 months of the study visit was recorded. Patients with UACR < 30mg/g were classified as normoalbuminuric, while patients with a UACR ≥ 30mg/g were classified as albuminuric.\n\nLevels of total CECs were measured by the Clinical Lab Services Group at Janssen Diagnostics, LLC (previously Veridex Corporation) using the CELLSEARCH® Circulating Endothelial Cell Kit (Janssen Diagnostics, LLC). This system has provided valid and reproducible CEC levels in normal populations and in patients with chronic illness and malignancy, and the details have previously been described in detail19,20. Briefly, peripheral whole blood was obtained with a 21-G butterfly needle and the first 3mL was discarded to minimize falsely elevated CEC levels related to venipuncture19. Whole blood was then collected into a tube containing anticoagulant and preservative, maintained at 15–30°C and shipped overnight to the central laboratory where it was processed within 72 hours. Prior studies have shown no significant difference in CEC levels at 0, 24, 48 and 72 hours in normal patients19. The CEC levels in a normal healthy population from this study are quoted from previous studies performed using the CELLSEARCH® Circulating Endothelial Cell Kit (Janssen Diagnostics, LLC)19.\n\nCECs were isolated and enumerated by Janssen Diagnostics, LLC using the CELLSEARCH® Circulating Endothelial Cell Kit (Janssen Diagnostics, LLC) in combination with the CELLTRACKS® AUTOPREP® System (Janssen Diagnostics, LLC), and the CELLTRACKS ANALYZER II® (Janssen Diagnostics, LLC). These systems use immunomagnetic bead technology and a semi-automated fluorescent microscope. Briefly, 4mL of blood were mixed with 10mL of dilution buffer containing 0.1% sodium azide and the mixture was centrifuged at 800g for 10min, placed in the sample preparation system, i.e. the CELLTRACKS® AUTOPREP® System (Janssen Diagnostics, LLC), and processed within 1 hour of preparation. The sample preparation system then aspirated the plasma, leaving the buffy coat layer and red cells to be further processed. An anti-CD146 ferrofluid from a 3.0mL suspension of 0.012% magnetic nanoparticles conjugated to a mouse monoclonal antibody was added (CELLSEARCH® Circulating Endothelial Cell Kit; Janssen Diagnostics, LLC). The cells were then incubated inside the sample preparation system at room temperature for 12 to 13 minutes, at which time they underwent magnetic separation. The unbound cells and remaining plasma were then aspirated by the sample preparation system, and staining reagents with a permeablization buffer, to fluorescently label immunomagnetically bound cells, were added. These staining reagents included: 1) 4,6-diamidino-2-phenylindole (DAPI), a nuclear stain, 2) the mouse monoclonal CD105-PE which is specific for the protein endoglin and is expressed by endothelial cells; activated monocytes, stromal cells and pre-B cells, and 3) the mouse monoclonal pan-leukocyte antibody CD45 conjugated to allophycocyanin, in conjuction with a permeabilization buffer (CELLSEARCH® Circulating Endothelial Cell Kit; Janssen Diagnostics, LLC). Cells were then re-incubated and magnetic separation was repeated. Finally, the cells were re-suspended in approximately 300 µL of buffer and transferred to a chamber with two magnets that magnetically oriented cells into a monolayer for analysis. Analysis was performed within 24 hours using the CELLTRACKS ANALYZER II (Janssen Diagnostics, LLC), a four-color semi-automated fluorescent microscope and a gray scale charged coupled device camera, capturing 175 frames per channel. The captured frames were analyzed by image analysis software. CECs candidates selected by the software were then manually identified by the operator as DAPI+, CD105+ and CD45- cells. A single CEC count was performed for each blood specimen.\n\nThe aim of this pilot trial was to study the feasibility of recruitment, and assessment procedures. The sample size of 40 was based on the feasibility of single center recruitment and the study budget. All normally distributed continuous variables are reported as mean±standard deviation, and all non-normally distributed variables as median (1st quartile – 3rd quartile). Categorical variables are reported as proportions. Kolmogorov-Smirnov test was used to evaluate the normality of all continuous variables. A dichotomous grouping variable was created using urine microalbumin variable (‘albuminuric’ if urine microalbumin >30 mg/g, ‘normo-albuminuric’ if <=30 mg/g). Two independent samples t-test was used to compare normally distributed variables between albuminuric and normo-albuminuric groups and Wilcoxon rank-sum test was used for non-normally distributed variables. Fisher’s exact test was used to compare all categorical variables. Correlations analyses between levels of CECs and other vascular injury markers were performed using Pearson’s correlation for normally distributed continuous variables and Spearman’s for non-normally distributed continuous variables. There were three missing data points in this study (von Willebrand factor antigen × 1, low density lipoprotein X 2). This missing data was ignored from analysis. All analyses were performed using SAS 9.4. Results were considered significant if p<0.05.\n\n\nResults\n\nThe characteristics of our study population are listed in Table 1. The average age of our group was 69.7 years, 52% were female, 72% were white and 20% were black (Table 1). A history of cardiovascular disease was present in 43% of participants, with a history of CAD in 35%, CHF in 8%, PVD in 10%, and stroke in 0%. Mean levels of all vascular injury markers were significantly higher than the reference range for our laboratory. Characteristics of albuminuric and normoalbuminuric subgroups are listed in Table 1. Only diabetes duration was significantly different between subgroups.\n\nNote. BMI, body mass index, DM, diabetes mellitus, CVD, cardiovascular disease, SBP, systolic blood pressure. DBP, diastolic blood pressure, eGFR, estimated glomerular filtration rate using the 4-variable modified diet in renal disease equation21, vWF, vonWillebrand factor, hsCRP, high sensitivity C-reactive protein, LDL, low-density lipoprotein. HbA1c, hemoglobin A1c\n\n†Variables are not normally distributed. Hence, comparisons were performed using Wilcoxon rank-sum test.\n\n‡p-values are from independent samples t-test for normally distributed continuous variables, Wilcoxon rank sum test for non-normally distributed variables and Fisher’s Exact test for categorical variables\n\nThe mean CEC level in our study patients was 38.7±38.1 cells, range 7–196 (Figure 1). These levels are significantly higher than levels tested in a historical normal population. In that study of 249 healthy subjects, done by Immunicon Corporation using the CELLSEARCH System®, the mean CEC level was 21±18 cells, range 0–9719. In our albuminuric subgroup, the average CEC level was 34.8±34.3 and in the normoalbuminuric subgroup the average CEC level was 42.9±42.5 (Figure 1). There was no significant difference in CEC levels between the albuminuric and normoalbuminuric groups (p=0.297).\n\nBox plots shows 25th, 50th (median) and 75th percentiles (horizontal bars). The lower fence is 1.5 × inter-quartile range (IQR) below the 25th percentile. The upper fence is 1.5 × IQR above the 75th percentile. The circle and plus signs inside the boxes are means. The circles outside the fences are outliers. Abbreviations: CEC, circulating endothelial cells; T2DM, type 2 diabetes mellitus; CKD, chronic kidney disease.\n\nTable 2 lists the correlation between CEC levels and other vascular injury markers tested in our study. We found a significant correlation between CEC levels and uric acid levels (r=0.33, p=0.039). No other significant correlations were found.\n\nNote. CEC, circulation endothelial cells, SBP, systolic blood pressure. DBP, diastolic blood pressure, BMI, body mass index, DM, diabetes mellitus, eGFR, estimated glomerular filtration rate using the 4-variable modified diet in renal disease equation (21), vWF, vonWillebrand factor, hsCRP, high sensitivity C-reactive protein. LDL, low-density lipoprotein. HbA1c, hemoglobin A1c. Hct, hematocrit\n\n†Non-normally distributed variables\n\n‡p-values are from Pearson correlation coefficient for normally distributed variables and Spearman correlation coefficient analyses for non-normally distributed variables\n\n\nDiscussion\n\nIn this study, we measured levels of CECs in patients with T2DM and CKD. We found that CEC levels are significantly higher than a normal population, and that there was no difference in these levels between normoalbuminuric and albuminuric subgroups. Additionally, CEC levels were significantly correlated with uric acid levels.\n\nCEC levels have been found to be elevated in patients with chronic kidney disease and patients that have received a kidney transplant. In a study of CEC levels in 29 patients undergoing hemodialysis, 10 patients with diabetes and stage 1 or 2 CKD, 7 patients with hypertension and stage 1 or 2 CKD and 22 healthy patients, Koc et al. found that CEC levels were significantly higher in the group on dialysis and in the groups with diabetes and hypertension compared to the healthy controls. Furthermore, these levels were significantly higher in dialysis patients with active compared to stable atherosclerotic cardiovascular disease18. We similarly found that CEC levels are higher in patients with T2DM and CKD when compared to CEC levels in a historical normal population. Additionally, our study is the first to demonstrate that CEC levels are elevated in normoalbuminuric patients with T2DM and CKD.\n\nOur finding of elevated CEC levels in normoalbuminuric patients suggests severe vascular injury despite normoalbuminuria. This finding has been seen in prior studies of patients with diabetes and CKD. Cross-sectional studies have shown no significant differences in the frequencies of diabetic retinopathy, coronary heart disease cerebrovascular disease or peripheral vascular disease between patients with normo, micro or macroalbuminuria and CKD7,14. Furthermore, renal biopsy studies of patients with T2DM, CKD and normoalbuminuria have revealed a higher prevalence of vascular injury compared to biopsies of patients with micro and macroalbuminuria22–24.\n\nOur results may have important clinical implications. We found a correlation between CEC levels and uric acid, a validated marker of vascular injury that has been linked to all-cause and cardiovascular mortality in patients with CKD25. This finding suggests that CEC levels may also provide information about future vascular risk in patients with T2DM and CKD, and we believe warrant future studies examining this association. Recent prospective studies have validated cerebral and renal ischemia, as measured by cerebral MRI and renal ultrasound, as biomarkers of future renal risk in patients with T2DM and CKD independent of urine microalbumin26,27. If larger studies confirm our findings, then a future prospective study testing the ability of CEC levels to predict renal and cardiovascular outcomes, even in normoalbuminuric CKD patients, may be warranted.\n\nOur pilot study is limited by its size and its cross-sectional design. Our goal for this study was to establish feasibility of patient recruitment and study procedures. Any observed associations need to be replicated in a larger-scale study, and none of the observed associations can be considered causal. Additionally, due to the small sample size, our results may not be applicable to the general CKD population.\n\nIn conclusion, we found that CEC levels are elevated in patients T2DM and CKD independent of urine microalbumin. These levels correlate with uric acid, a known marker of vascular injury. Larger, prospective studies are needed to confirm these findings.\n\n\nData availability\n\nHarvard Dataverse: Dataset. Shirazian, Shayan, 2016, “Replication Data for: A pilot trial to characterize circulating endothelial cells in patients with type 2 diabetes and chronic kidney disease”, http://dx.doi.org/10.7910/DVN/I64SAC28\n\n\nConsent\n\nWritten informed consent for publication of their clinical details was obtained from all participants.", "appendix": "Author contributions\n\n\n\nS.S. conceived the study, obtained funding for the study, designed the experiments, prepared the data for analysis, wrote the manuscript, and reviewed and edited the manuscript. C.G., V.R., R.S., R.P. helped recruit participants for the study, collected study data, and reviewed and edited the manuscript. S.I. prepared the data for analysis, carried out all analyses and reviewed and edited the manuscript. J.M. helped conceive the study and reviewed and edited the manuscript. All authors take responsibility for the integrity and accuracy of the data, were involved in revising a draft of the manuscript and agree to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study was supported by a Winthrop University Hospital pilot grant awarded to S.S.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe would like to thank Robert Diep, Monika Wadhwani, Marilyn Richardson, and Maung Aung for helping to recruit study participants and collect study data.\n\n\nReferences\n\nCoresh J, Selvin E, Stevens LA, et al.: Prevalence of chronic kidney disease in the United States. JAMA. 2007; 298(17): 2038–2047. PubMed Abstract \| Publisher Full Text\n\nUnited States Renal Data System: 2015 USRDS annual data report: Epidemiology of kidney disease in the United States. National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD, 2015. Reference Source\n\nMolitch ME, DeFronzo RA, Franz MJ, et al.: Nephropathy in diabetes. Diabetes Care. 2004; 27(Suppl 1): S79–83. PubMed Abstract \| Publisher Full Text\n\nKramer H, Molitch ME: Screening for kidney disease in adults with diabetes. Diabetes Care. 2005; 28(7): 1813–1816. PubMed Abstract \| Publisher Full Text\n\nChen YH, Chen HS, Tarng DC: More impact of microalbuminuria on retinopathy than moderately reduced GFR among type 2 diabetic patients. Diabetes Care. 2012; 35(4): 803–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKramer HJ, Nguyen QD, Curhan G, et al.: Renal insufficiency in the absence of albuminuria and retinopathy among adults with type 2 diabetes mellitus. JAMA. 2003; 289(24): 3273–3277. PubMed Abstract \| Publisher Full Text\n\nMacIsaac RJ, Tsalamandris C, Panagiotopoulos S, et al.: Nonalbuminuric renal insufficiency in type 2 diabetes. Diabetes Care. 2004; 27(1): 195–200. PubMed Abstract \| Publisher Full Text\n\nPenno G, Solini A, Bonora E, et al.: Clinical significance of nonalbuminuric renal impairment in type 2 diabetes. J Hypertens. 2011; 29(9): 1802–1809. PubMed Abstract \| Publisher Full Text\n\nRetnakaran R, Cull CA, Thorne KI, et al.: Risk factors for renal dysfunction in type 2 diabetes: U.K. Prospective Diabetes Study 74. Diabetes. 2006; 55(6): 1832–1839. PubMed Abstract \| Publisher Full Text\n\nYokoyama H, Sone H, Oishi M, et al.: Prevalence of albuminuria and renal insufficiency and associated clinical factors in type 2 diabetes: the Japan Diabetes Clinical Data Management study (JDDM15). Nephrol Dial Transplant. 2009; 24(4): 1212–1219. PubMed Abstract \| Publisher Full Text\n\nAraki S, Haneda M, Sugimoto T, et al.: Factors associated with frequent remission of microalbuminuria in patients with type 2 diabetes. Diabetes. 2005; 54(10): 2983–2987. PubMed Abstract \| Publisher Full Text\n\nKrolewski AS: Progressive renal decline: the new paradigm of diabetic nephropathy in type 1 diabetes. Diabetes Care. 2015; 38(6): 954–962. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThorn LM, Gordin D, Harjutsalo V, et al.: The Presence and Consequence of Nonalbuminuric Chronic Kidney Disease in Patients With Type 1 Diabetes. Diabetes Care. 2015; 38(11): 2128–33. PubMed Abstract \| Publisher Full Text\n\nIto H, Takeuchi Y, Ishida H, et al.: High frequencies of diabetic micro- and macroangiopathies in patients with type 2 diabetes mellitus with decreased estimated glomerular filtration rate and normoalbuminuria. Nephrol Dial Transplant. 2010; 25(4): 1161–1167. PubMed Abstract \| Publisher Full Text\n\nRabelink TJ, de Boer HC, van Zonneveld AJ: Endothelial activation and circulating markers of endothelial activation in kidney disease. Nat Rev Nephrol. 2010; 6(7): 404–414. PubMed Abstract \| Publisher Full Text\n\nBlann AD, Woywodt A, Bertolini F, et al.: Circulating endothelial cells. Biomarker of vascular disease. Thromb Haemost. 2005; 93(2): 228–235. PubMed Abstract \| Publisher Full Text\n\nBoos CJ, Lip GY, Blann AD: Circulating endothelial cells in cardiovascular disease. J Am Coll Cardiol. 2006; 48(8): 1538–1547. PubMed Abstract \| Publisher Full Text\n\nKoç M, Bihorac A, Segal MS: Circulating endothelial cells as potential markers of the state of the endothelium in hemodialysis patients. Am J Kidney Dis. 2003; 42(4): 704–712. PubMed Abstract \| Publisher Full Text\n\nRowand JL, Martin G, Doyle GV, et al.: Endothelial cells in peripheral blood of healthy subjects and patients with metastatic carcinomas. Cytometry A. 2007; 71(2): 105–113. PubMed Abstract \| Publisher Full Text\n\nCristofanilli M, Budd GT, Ellis MJ, et al.: Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med. 2004; 351(8): 781–791. PubMed Abstract \| Publisher Full Text\n\nLevey AS, Bosch JP, Lewis JB, et al.: A more accurate method to estimate glomerular filtration rate from serum creatinine: a new prediction equation. Modification of Diet in Renal Disease Study Group. Ann Intern Med. 1999; 130(6): 461–470. PubMed Abstract \| Publisher Full Text\n\nEkinci EI, Jerums G, Skene A, et al.: Renal structure in normoalbuminuric and albuminuric patients with type 2 diabetes and impaired renal function. Diabetes Care. 2013; 36(11): 3620–3626. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoriya T, Suzuki Y, Inomata S, et al.: Renal histological heterogeneity and functional progress in normoalbuminuric and microalbuminuric Japanese patients with type 2 diabetes. BMJ Open Diabetes Res Care. 2014; 2(1): e000029. PubMed Abstract \| Publisher Full Text\n\nShimizu M, Furuichi K, Yokoyama H, et al.: Kidney lesions in diabetic patients with normoalbuminuric renal insufficiency. Clin Exp Nephrol. 2014; 18(2): 305–312. PubMed Abstract \| Publisher Full Text\n\nMadero M, Sarnak MJ, Wang X, et al.: Uric acid and long-term outcomes in CKD. Am J Kidney Dis. 2009; 53(5): 796–803. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUzu T, Kida Y, Shirahashi N, et al.: Cerebral microvascular disease predicts renal failure in type 2 diabetes. J Am Soc Nephrol. 2010; 21(3): 520–526. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNosadini R, Velussi M, Brocco E, et al.: Increased renal arterial resistance predicts the course of renal function in type 2 diabetes with microalbuminuria. Diabetes. 2006; 55(1): 234–239. PubMed Abstract \| Publisher Full Text\n\nShirazian S: Replication Data for: A pilot trial to characterize circulating endothelial cells in patients with type 2 diabetes and chronic kidney disease. Harvard Dataverse, V1 [UNF:6:g4naiki55I5odFzgsSXdqw==]. 2016. Data Source" }	[ { "id": "13202", "date": "06 Apr 2016", "name": "Albert Dreisbach", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nNovel pilot study demonstrating significant increase in circulating vascular endothelial cells (CEC) in stage 3 chronic kidney disease with and without microalbuminuria suggesting significant vascular disease in patients with normal albuminuria and albuminuria compared to historical healthy controls. Significant correlation was also shown between CEC and serum uric acid. This is a preliminary study and prospective outcome studies will need to be performed to demonstrate any clinical predictive power of CECs. This limitation the investigators acknowledge.It would be interesting to measure CEC levels in T2DM without CKD (normal eGFR without microalbuminuria) and follow them prospectively to see whether they are predictive of development of CKD.", "responses": [] }, { "id": "14122", "date": "02 Jun 2016", "name": "Bernard Canaud", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis pilot study was performed in 40 patients type 2 diabetes mellitus (T2DM) and stage 2 chronic kidney disease (CKD) and examine the association between circulating endothelial cell (CEC) levels and surrogates biomarkers of vascular damage (urine albumin, von-Willebrand factor antigen, hs-CRP, uric acid). Mean CEC levels were significantly higher (x1.8) than the normal population independently for albuminuria level and correlated with uric acid levels. Higher CEC levels have been already reported in severe renal disease (T1 and T2DM, CKD, Lupus…) and cardiovascular disease (ischemic cardiac disease, angioplasty coronary stenting or bypassing). CEC are considered as markers of severe vascular damage with poor outcome.\n\nThe originality of this study is to identify that CEC levels are elevated also in normoalbuminuric patients with T2DM and CKD suggesting severe vascular injury despite the absence of significant albuminuria.\nUnfortunately, the predictive value of CEC levels either on cardiovascular disease or chronic kidney disease progression is missing. In addition, it would have been interesting to have data on endothelial progenitor cell (EPC) levels, which are markers of endothelial regeneration with vascular protective role.\n\nAs the authors mentioned, further prospective studies are needed to define the role and the predictive value of CEC in T2DM and CKD patients as biomarker of cardiovascular and kidney disease progression.", "responses": [] } ]	1	https://f1000research.com/articles/5-292
https://f1000research.com/articles/5-290/v1	07 Mar 16	{ "type": "Case Report", "title": "Case Report: A giant but silent adrenal pheochromocytoma – a rare entity", "authors": [ "Sunil Munakomi", "Saroj Rajbanshi", "Prof Shailesh Adhikary", "Saroj Rajbanshi", "Prof Shailesh Adhikary" ], "abstract": "Herein we report a rare entity of a giant adrenal pheochromocytoma in a fifty-year-old male presenting with a vague abdominal pain. A computerised tomogram of the abdomen revealed a well-defined left supraadrenal giant lesion with no evidence of invasion to surrounding structures.The patient underwent surgical excision without any untoward postoperative events. Histopathological study revealed a benign pheochromocytoma. This report highlights the importance of acknowledging the fact that sometimes a giant adrenal pheochromocytoma can present with paucity of clinical signs and symptoms.Thorough investigations and a multidisciplinary team approach may lead to a better outcome in these patients.", "keywords": [ "Giant", "benign", "silent", "pheochromocytoma" ], "content": "Introduction\n\nGiant pheochromocytomas (> 7 cm in size) are rare entities with around 20 cases reported in the literature1–6. They do not present with the classical symptoms of pheochromocytomas6. Most patients present with vague discomfort while others may complain of a palpable abdominal mass. Operative surgery is the ideal management option7. There needs to be a multidisciplinary approach while managing such cases. Stringent preparation to combat crisis due to catecholamine surge (during tumor manipulation) and sudden decrease in peripheral vascular resistance (following lesional excision) need to be emphasized8,9. Presence of chromaffin cells in the extra-adrenal tissue is the only confirmative method of distinguishing the malignant variant from its benign counterpart10. Herein we highlight and discuss the management algorithm taken while managing one such case.\n\n\nCase report\n\nA fifty-year-old male from Dhahran, Nepal presented to the surgical outpatient clinic with a vague symptom of abdominal discomfort. He had no history of trauma, persistent vomiting, altered bowel habits, change in the color of the stool or abdominal distension. There were no significant past medical or surgical illnesses. Family history of similar symptoms was also absent. Examination of the abdomen was normal except for slight discomfort during palpation in his left upper quadrant. Ultrasound examination of the abdomen revealed a huge left suprarenal mass. Computerised tomogram (CT) of the abdomen confirmed a giant mass of approximately 12×8 cm2 in the left suprarenal region showing rim enhancement and areas of low attenuation within it. The left renal vein was normal and the lesion was slightly abutting the spleen (Figure 1).\n\nThe patient denied attacks of headache, cheat pain, palpitation and sweating. The serum and urinary catecholamine levels were within normal range. The patient was kept for observation with 24 hour electrocardiography (ECG) and blood pressure monitoring (BP) which was normal.\n\nThe patient and his relatives were explained of the disease entity and were advised for surgery. With written consent, he was prepared for surgery. The anesthesiologists prepared medication (Intravenous (i.v.) Phentolamine (1 mg injection), Nitroprusside (4 mg drip) and Esmolol (30 mg injection) for potential intra-operative crisis pertaining to catecholamine surge during surgical manipulation. Vasoactive agents were also made available for combating sudden loss of peripheral vascular resistance following tumor removal. Early vascular control was secured. There was a well demarcated plane to dissect the tumor from the surrounding structures (Figure 2).\n\nThe patient was extubated and was kept in the intensive care unit (ICU) for 48 hours. There were no untoward events in the post-operative period and the patient was discharged home on the 10th day. Histopathological study revealed zellballen nests of chromaffin cells with no invasion of the capsule (Figure 3), which is highly suggestive of a benign pheochromocytoma.\n\nThe patient is asymptomatic 4 years following surgery, and has been advised to follow up periodically in order to rule out early recurrence.\n\n\nDiscussion\n\nPheochromocytomas typically present with the characteristic triad of paroxysmal attacks of headache, sweating and palpitation11. However giant lesions paradoxically may lack these symptoms6. The reasons for the same can be due of the presence of tumoral necrosis, high loads of interstitial tissue compared to chromaffin cells or the paucity of the release of the catecholamines due to encapsulation by the connective tissues6. This may also cause normal catecholamine values during their serum and urinary assays2.\n\nCT scan is the image modality of choice to diagnose the condition4. However, in cases of giant lesions, there may be difficulties indetermining the organ of origin o leading to mis-diagnosis of the entity2.\n\nOpen surgical removal is the therapeutic target7. Laparoscopic removal is reserved only for smaller lesions12,13. Some authors have suggested preoperative embolisation of theses lesion14. However, it may be tenacious due to major arterio-venous connections within the lesion6. The key to a successful outcome is the fine tuning between the surgeons and the anesthesist in the peri-operative period8,9. There needs to be minimal handling of the lesion and an early control of the adrenal vein so as to limit crisis due catecholamine surge6.\n\nPheochromocytoma of the adrenal gland scaled score (PASS) score has been described to differentiate between the benign and the malignant lesions15. But the hallmark of the malignant counterpart is the presence of the ectopic chromaffin cells in the extra-adrenal sites10.\n\nThe patients need to be on a periodic follow up so as to exclude the risk of recurrence16. There are still no set therapeutic guidelines in the management of the malignant lesions due to paucity of cases. Long term prognosis is dismal with five year survival of around 50% only17.\n\n\nConclusion\n\nThough benign, surgery is advocated for giant pheochromocytomas. Early vascular control, minimal handling of the tumor and a multidisciplinary approach to combat potential intra-operative crisis are the cornerstones in managing such cases. Malignant counterparts need to be excluded histologically. Patients require regular follow up to rule out recurrence.\n\n\nConsent\n\nWritten informed consent was obtained from the patient for publication of this case report and any accompanying images and/or other details that could potentially reveal the patient’s identity.", "appendix": "Author contributions\n\n\n\nDr Sunil prepared the manuscript and obtained the pictures. Dr Saroj and Prof Shailesh revised and confirmed the final manuscript. All authors have seen and agreed to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nThe authors declare no conflict of interest.\n\n\nGrant information\n\nThe authors declare that no funding was involved in supporting this work.\n\n\nReferences\n\nSarveswaran V, Kumar S, Kumar A, et al.: A giant cystic pheochromocytoma mimicking liver abscess an unusual presentation - a case report. Clin Case Rep. 2015; 3(1): 64–68. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang HL, Sun BZ, Xu ZJ, et al.: Undiagnosed giant cystic pheochromocytoma: A case report. Oncol Lett. 2015; 10(3): 1444–1446. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKorgali E, Dundar G, Gokce G, et al.: Giant malignant pheochromocytoma with palpable rib metastases. Case Rep Urol. 2014; 2014: 354687. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAmbati D, Jana K, Domes T: Largest pheochromocytoma reported in Canada: A case study and literature review. Can Urol Assoc J. 2014; 8(5–6): E374–E377. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSoufi M, Lahlou MK, Benamr S, et al.: Giant malignant cystic pheochromocytoma: a case report. Indian J Surg. 2012; 74(6): 504–506. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi C, Chen Y, Wang W, et al.: A case of clinically silent giant right pheochromocytoma and review of literature. Can Urol Assoc J. 2012; 6(6): E267–E269. PubMed Abstract \| Free Full Text\n\nStaren ED, Prinz RA: Selection of patients with adrenal incidentalomas for operation. Surg Clin North Am. 1995; 75(3): 499–509. PubMed Abstract\n\nAdenekan A, Faponle A, Badmus T, et al.: Anaesthetic management of giant phaeochromocytoma in a patient with chronic renal disease. J West Afr Coll Surg. 2011; 1(2): 112–122. PubMed Abstract \| Free Full Text\n\nAntedomenico E, Wascher RA: A case of mistaken identity: giant cystic pheochromocytoma. Curr Surg. 2005; 62(2): 193–198. PubMed Abstract \| Publisher Full Text\n\nGlodny B, Winde G, Herwig R, et al.: Clinical differences between benign and malignant pheochromocytomas. Endocr J. 2001; 48(2): 151–9. PubMed Abstract \| Publisher Full Text\n\nRodríguez González JM, Parrilla Paricio P, Piñero Madrona A: Feocromocitoma. In: Sitges-Serra A, Sancho Insenser J, editors. Cirugía Endocrina. Madrid, Spain: Aran, 1999; 143–150.\n\nWang HS, Li CC, Chou YH, et al.: Comparison of laparoscopic adrenalectomy with open surgery for adrenal tumors. Kaohsiung J Med Sci. 2009; 25(8): 438–44. PubMed Abstract \| Publisher Full Text\n\nTiberio GA, Baiocchi GL, Arru L, et al.: Prospective randomized comparison of laparoscopic versus open adrenalectomy for sporadic pheochromocytoma. Surg Endosc. 2008; 22(6): 1435–9. PubMed Abstract \| Publisher Full Text\n\nWang D, Li H, Zhang Y, et al.: Selective arterial embolization in giant pheochromocytoma. Zhong Hua Mi Niao Wai Ke Za Zhi. 2011; 32(5): 299–302. Publisher Full Text\n\nThompson LD: Pheochromocytoma of the Adrenal gland Scaled Score (PASS) to separate benign from malignant neoplasms: a clinicopathologic and immunophenotypic study of 100 cases. Am J Surg Pathol. 2002; 26(5): 551–566. PubMed Abstract\n\nLow G, Sahi K: Clinical and imaging overview of functional adrenal neoplasms. Int J Urol. 2012; 19(8): 697–708. PubMed Abstract \| Publisher Full Text\n\nEisenhofer G, Bornstein SR, Brouwers FM, et al.: Malignant pheochromocytoma: current status and initiatives for future progress. Endocr Relat Cancer. 2004; 11(3): 423–36. PubMed Abstract \| Publisher Full Text" }	[ { "id": "13171", "date": "22 Apr 2016", "name": "Venugopal Sarveswaran", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 feel the case report seems to be an original one, though such cases have been published previously. It provides sufficient details for others to learn, like it emphasizes that such clinical condition must be kept in mind and adequate safety precautions must be taken while operating on such patients, though all cases will not have intra-operative complications. It states the clinical condition and investigations done. An adequate number of references have been sited. Hence I feel the article is acceptable.", "responses": [] }, { "id": "16138", "date": "12 Sep 2016", "name": "Prasanna Kumar Reddy", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe format of the paper and the language are good. Giant adrenal tumors are not that rare, but always pose a diagnostic dilemma.\nThe CT pictures are informative and remaining diagnostic work up was done well. Even though it is solitary, non functioning, and showing no evidence of capsular invasion or any distant metastasis, a lot of emphasis was given on malignancy. The size of the tumor does not have any significance on malignancy. However one has to be aware of malignant potential.\nThe technique was described well, but the importance of not breaching the capsule was not sufficiently emphasized. Intraoperative hemodynamic parameters were not highlighted.\nPresently the procedure of choice is laparoscopic adrenalectomy even up-to the size of 12 cm in minimally invasive surgical units with expertise as left supra renal is easily amenable. Vascular control with the presently available gadgets is much easier and the manipulation of the tumors is very minimal.\nCorrections:\nAbstract - supra adrenal gland was mentioned instead of supra renal gland.\n\nCase report - Line 9 instead of chest pain it is misspelled as cheat pain.\nThe conclusion, even though it is adequate, is too brief.\nAn adequate number of references were quoted.\nImpression - A good paper contributing various facts of giant pheochromocytoma, even though it is a single case report.", "responses": [] } ]	1	https://f1000research.com/articles/5-290
https://f1000research.com/articles/5-280/v1	04 Mar 16	{ "type": "Opinion Article", "title": "Communities of practice in life sciences and the need for brokering", "authors": [ "Anne Tierney" ], "abstract": "Etienne Wenger’s work on communities of practice is influential in teaching and learning in higher education. A core work of many postgraduate certificate in teaching and learning (PGCert) courses for new lecturers, it is studied, in the main, as a means to understand how to support and encourage students to achieve more effective learning. Communities of practice can also be applied to academics. In the context of the Research Excellence Framework (REF) and its predecessors, the gulf between research-focused and teaching-Focused academics in life sciences has widened, so that in many institutions, these two groups have evolved into two distinct communities of practice; one whose priority is disciplinary research, the other’s learning and teaching. However, in 2015, the UK government announced that a Teaching Excellence Framework (TEF) would be introduced into higher education in England, as early as 2017. While the exact details of TEF remain unclear, it is certain that “excellence” and “student satisfaction” will be high on the agenda. It is vital, therefore, that the two communities of practice, research-focused and teaching-focused, find ways to come together in order to ensure high quality teaching and learning. Wenger proposes that this can be done through the process of “brokering”, which allows expertise from both communities of practice to cross from one to the other, strengthening both. This should be encouraged at departmental and institutional level, but another vital origin of brokering can be forged at a(n) (inter)national level at meetings such as the SEB Annual Conference, where teaching-focused academics have the opportunity to mix with research-active colleagues. While this paper is informed by recent and current events in the UK Higher Education sector, it is of interest to academics who work in an environment where research and teaching have become separate to any extent.", "keywords": [ "Community of Practice", "brokering", "collaboration", "teaching and learning", "pedagogy" ], "content": "Introduction\n\nThis paper is based on my experiences as a life sciences teaching-focused academic in a research intensive UK university, and my experiences working with organisations such as The Higher Education Academy, Quality Assurance Agency (Scotland) and The Society for Experimental Biology’s SEB+. It is also based on my observations of colleagues, and is related to my current doctoral work, although this paper represents a more general form of themes which have arisen from the study. The opinions contained in the paper are my own, and are influenced by the work of Etienne Wenger and Torgny Roxå and his colleagues. As such, my opinions and interpretations are open to discussion, and I welcome others’ thoughts on this subject.\n\n\nCommunities of practice: focus on academics\n\nEtienne Wenger’s work on communities of practice (1998) is influential in teaching and learning in higher education. It is used, primarily, to support student learning communities of practice, promoting the idea that learning is a social endeavour (Bandura, 1977; Engeström, 1987; Parsons, 1962; Vygotsky, 1934). However, Wenger’s work can also be used to investigate how academics form communities of practice, the dimensions of which are defined by (1) mutual engagement; (2) joint enterprise; and (3) a shared repertoire (Wenger, 1998, p. 73). In the UK, a number of external pressures, such as the Research Excellence Framework (Higher Education Funding Council for England) and National Student Survey has increasingly meant the separation of research-focused and teaching-focused activities, resulting in the emergence of two distinct communities of practice; one whose enterprise priority is disciplinary research, the other, teaching and learning. The separation of joint enterprise coincides with differences in engagement and repertoire. Differences in engagement manifest as a withdrawal of research-focused academics from teaching, while teaching-focused academics reduce their involvement in, or are not required to engage in disciplinary research. Differences in repertoire can be seen as teaching-focused academics develop their expertise in the Scholarship of Teaching and Learning (SoTL), while research-focused academics develop their disciplinary expertise. Thus, the two groups diverge, forming two distinct communities of practice.\n\n\nResearch-focused or teaching-focused: establishment of two communities of practice\n\nAcademics’ initial introduction to Wenger’s work is generally through the PGCert, which, post Dearing Report (1997) most institutions now require probationers to complete, although the absolute requirements for completion vary a great deal (Gosling, 2010). At this point, individuals, although set on course to become members of either the research-focused or teaching-focused community of practice, are required to go through academic probation, which includes an introductory qualification in teaching and learning (PGCert). Completion of the PGCert, under its many guises, marks a departure point for academics; for many, it marks the end of a compulsory period of enquiry into pedagogy, and once the PGCert has been obtained, the business of establishing one’s disciplinary research reputation takes priority. For others, it marks the beginning of development of expertise in pedagogy, and the following of a teaching and scholarship career path. Thus, the two communities of practice diverge at this point. The divergence is aided by the pressures of the REF, which places emphasis on the production of world class disciplinary research (Higher Education Funding Council for England), as well as differentiated career routes for staff (for example, the University of Glasgow has four academic career routes, designated “Research & Teaching”, “Research”, “Teaching & Scholarship” and “Veterinary Clinician”, all of which go as far as Professor (‘Human Resources, College of MVLS’)).\n\nWhile the separation of academics into research-focused and teaching-focused has its advantages, for example, allowing research-focused academics to concentrate on disciplinary research, while teaching-focused academics concentrate on teaching and learning, there are also disadvantages, research-focused academics do not develop their expertise in teaching and learning beyond the basics they are introduced to during their probationary period, and, while research-focused academics are still required to teach, this happens, in the main, in the form of didactic lectures. Teaching-focused academics, on the other hand, are required to develop their expertise in pedagogy, and are more inclined to introduce a variety of student-centred, active learning activities into their teaching, which supports student learning. However, this comes at the cost of developing their disciplinary expertise, as they leave active disciplinary research for pedagogy. Therefore there is a danger which impacts on the “student experience” – Research-focused academics whose disciplinary expertise is world class, but whose pedagogic knowledge is basic, and teaching-focused academics whose pedagogic expertise has been developed, but whose disciplinary expertise is hampered by no longer being research-active. While this has minimal impact on early years undergraduate education, it may impact on later years, in particular with undergraduate research projects, or with Masters level research projects. This situation has perpetuated over a number of years. However, there is a new influence coming to the higher education landscape, of England in particular, which may affect the two communities of practice, and, depending on institutions’ responses to it, has the potential to further divide the communities of practice, or act as a catalyst to unite them. This new influence is the Teaching Excellence Framework (Johnson, 2015) which is proposed to be imposed on higher education in England as early as 2017. Despite education being a devolved matter, the influence of TEF is likely to be felt within institutions in all four home nations. While the details of the implementation of TEF remain unknown, the Green Paper sets great store in “excellence” and “student experience”, with one proposal being the introduction of an “Office for Students” (Johnson, 2015, p. 62), to supersede The Quality Assurance Agency for Higher Education (QAA), one of its powers being “[to ensure] the rights of students to hold providers to account and get value for money for their investment, and to protect them in the event of a provider exiting the sector”. It is to be anticipated, therefore, that institutions will be subject to market forces, and students will be encouraged to seek “satisfaction”. Therefore, it is necessary to somehow realign the joint ventures of research-focused and teaching-focused academics in order to provide a research-led experience for students.\n\n\nBrokering: making connections across the boundaries\n\nWenger’s (1998) communities of practice do not operate in isolation. In reality, while communities of practice are distinct and can be identified as such, they work alongside one another, and mutually benefit from connections which are made across the boundaries between them. Connections between communities of practice are made in two ways, either via boundary objects or by brokering. Boundary objects are defined as “artifacts, documents, terms, concepts and other forms of reification around which communities of practice can organize their interconnections”, while brokering is defined as “connections provided by people who can introduce elements of one practice into another” (Wenger, 1998, p. 105). As it stands, the research-focused and teaching-focused communities of practice share many boundary objects; a common knowledge base, common vocabulary and discourse, common or similar environments, equipment, methodologies. All of these serve as connections between the research-focused and teaching-focused communities of practice, identifying the participants as life scientists, albeit with a different primary focus of activities. In contrast, the opportunities for brokering remain limited, and the individuals who participate in the communities of practice remain, by and large, separated by the structures of the institution. Given that there are many boundary objects which the two communities of practice share, opportunities for brokering could be better facilitated. Opportunities to improve brokering necessitate the recognition of two distinct boundary objects which can be used as capital to facilitate exchange: The Scholarship of Teaching and Learning (SoTL), owned by the teaching-focused community and up-to-date Disciplinary Research, owned by the research-focused Community.\n\n\nAdvantages of fostering brokering relationships between communities\n\nBrokering relationships between teaching-focused academics and research-focused academics works to the advantage of both communities. While it would be naïve to claim that these relationships do not presently exist, it is my experience that they are neither common, nor long-lasting, but rather exist when convenient, on an ad hoc basis, for example, when a particular project or initiative is launched. However, given the proposals of the TEF to foreground teaching and learning and redress the imbalance between it and research, it becomes necessary to facilitate these brokering relationships in a more sustainable fashion. Thus, research-focused academics bring with them their latest research findings with which to inform an undergraduate learning activity, while teaching-focused academics bring the pedagogic underpinnings with which to make the learning experience valuable to the students. Simultaneously, as each brings their own expertise to the relationship, they also learn from one another, working at the periphery of each other’s expertise. This can work at the level of individual academics, working within a department, and at any level; postgraduate, postdoctoral fellow, probationer, lecturer, professor. Teaching-focused academics benefit from this arrangement by working with colleagues who have current research knowledge, while research-focused academics learn techniques which they can use to improve their teaching. The benefits go beyond the immediate exchange of expertise; graduate students who participated in undergraduate teaching saw improvements in their research methodological skills (Feldon et al., 2011). It may also be possible to extend this exchange by accommodating teaching-focused academics in research labs where they could update their research skills, improving their currency for subsequent years.\n\n\nSeeking excellence in teaching and learning\n\nGiven the competing pressures that academics work under, it is understandable why individuals may be sceptical of yet more work. However, the work of Roxå and colleagues (Roxå & Mårtensson, 2011; Roxå et al., 2007) suggests that there are two strategies which departments employ when attempting to improve “excellence” in teaching and learning, one of which is more likely to be more successful than the other. In what they term “Trajectory 1”, there are a few experts in pedagogy within a department who are relied upon by the colleagues to implement teaching and learning innovations. However, this leads to patchy application of pedagogic principles, which translates into differences in student experience. Roxå and his colleagues suggest that “Trajectory 2” represents a better strategy for departmental excellence in teaching and learning, by having every academic participate in it to a greater or lesser extent, thereby affecting departmental culture. In order for this to happen, it is necessary to broker relationships between Teaching-focused academics, who already have pedagogic knowledge, and research-focused academics, who have disciplinary research knowledge.\n\n\nThe role of learned societies\n\nBrokering also has the potential to work at a broader level, between institutions. In order for brokering relationships to be facilitated and encouraged, it is vital to have individuals from both communities of practice mixing together, and communicating their expertise. For life scientists, meetings such as those organised by learned societies are vital as a means for research-focused and teaching-focused academics to mix. For example, the SEB+ session at the 2015 SEB Annual Conference in Prague was a vibrant, well-attended day of presentations and discussion, notable for the mix of research-focused and teaching-focused academics in attendance. Plans are already in place for another SEB+ session in Brighton in 2016, based on its success in Prague. The SEB+ session offers a space where the issues of teaching and learning in a scholarly manner can be discussed in a supportive environment, and where good practice can be shared. While it is possibly too early to draw any conclusions from the session, if it gives research-focused academics an opportunity to reflect on their teaching practice, and sparks some level of interest in pedagogy, then it is to be encouraged, and accepted as a regular part of the conference programme.\n\n\nConclusions\n\nDisciplinary research has enjoyed a prioritisation within UK Higher Education, fuelled by the pressures of the REF. While the proposed TEF seeks to address this imbalance, and restore the status of teaching and learning in higher education, at this time it does not propose how it will do it. This paper has laid out a proposal, underpinned by pedagogic research theory, which suggests that closer working relationships between teaching-focused and research-focused academics is necessary in order to address “excellence”, by developing the disciplinary and pedagogic expertise of academics by facilitating the exchange of knowledge via expert relationships, which Wenger names “brokering”. The opportunities for brokering may take place within a department, but there is also a role for learned societies, such as The Society for Experimental Biology, to support brokering relationships between disciplinary and pedagogic practitioners.\n\nI welcome discussion on this topic.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author declared that no grants were involved in supporting this work.\n\n\nReferences\n\nBandura A: Social learning theory. Eaglewood Cliffs, NJ, Prentice-Hall, 1977; 247. Reference Source\n\nDearing R: The National Committee of Inquiry into Higher Education. 1997. Reference Source\n\nEngeström Y: Learning by expanding: An activity-theoretical approach to developmental research. Helsinki: Orienta-Konsultit, 1987. Reference Source\n\nFeldon DF, Peugh J, Timmerman BE, et al.: Graduate students’ teaching experiences improve their methodological research skills. Science. 2011; 333(6045): 1037–1039. PubMed Abstract \| Publisher Full Text\n\nGosling D: Is your Pg Cert compulsory for new staff? A summary of responses from the SEDA JISCMail list. 2010. Reference Source\n\nHigher Education Funding Council for England: (n.d.). The Research Excellence Framework. 2013. Reference Source\n\nHuman Resources, College of MVLS. (n.d.), 2015. Reference Source\n\nJohnson J: Fulfilling our potential: Teaching Excellence, Social Mobility and Student Choice. Cm9141, 2015. Reference Source\n\nParsons T: The Structure of Social Action. New York: Free Press, 1962.\n\nRoxå T, Mårtensson K: Understanding strong academic microcultures - An exploratory study. University of Lund, 2011. Reference Source\n\nRoxå T, Olsson T, Mårtensson K: Scholarship of Teaching and Learning as a strategy for institutional change. In Proceedings of the 30th HERDSA Annual Conference. Adelaide, South Australia: Higher Education Research and Development Society of Australasia, Inc., 2007; 487–494. Reference Source\n\nThe National Student Survey. (n.d.). Reference Source\n\nVygotsky LS: Thought and Language. Cambridge, MA: MIT Press, 1934. Reference Source\n\nWenger E: Communities of Practice: Learning, Meaning and Identity. Cambridge University Press, 1998. Reference Source" }	[ { "id": "12754", "date": "08 Mar 2016", "name": "Peter Klappa", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI think this is a very well written opinion article, which is underpinned by the relevant research in Higher Education, especially pertinent to the work of Eric Wenger et al. The concept of 'brokering' to bridge the gap between the different communities of practice is very important, especially in a time of tension between research and teaching. It will be very interesting to see, how this concept is transformed into reality through emerging examples and case studies.The article should be indexed as is without any further corrections.", "responses": [] }, { "id": "12751", "date": "14 Mar 2016", "name": "Kevan M.A. Gartland", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis Opinion Article raises a number of very interesting points about the inter-relationship between research academic specialists and those pursuing a learning and teaching led specialism. It highlights the tensions that may frequently emerge between the two and proposes the use of brokering as a way forward, in the context of the emergent Teaching Excellence Framework (TEF), for universities in England, which will also have impacts and change behaviours in the rest of the UK and further afield. The author emphasises the value of enhancing relationships between these two communities of practice, and the role that Learned Societies, for example, can play to promote greater interchange of ideas and experiences between these groups. Without such initiatives, these groups which may also extend beyond single departments and across whole institutions, may diverge further leading to a paucity of student learning experience and the detriment of lost knowledge, skills and expertise that might not meet institutional Research Excellence Framework priorities. The rise of the TEF provides an opportunity for the imbalance of priorities between research and learning & teaching which may arise, to be redressed for the benefit of all. The article explains the opinions stated well, placing them into the context of previous work. The conclusions are balanced and justified. The article is acceptable for approval, subject to the following minor corrections: 'teaching-focused' (Abstract, line 7 of pdf), 'community' (lower case 'c'; at end of para 1 page 4 of pdf), and 'teaching-focused' (para 3, page 4 of pdf).", "responses": [] }, { "id": "12753", "date": "17 Mar 2016", "name": "Morven C. Shearer", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 thoughtful and timely piece, looking at the different communities of practice (CoPs) that exist in our HEIs currently and arguing that these communities must work together (despite the pressures of REF and, the potential-TEF, drawing them apart) to achieve excellence in teaching and learning. I think Tierney has raised good points, grounded in the literature around CoPs, concerning the need for 'brokering relationships' and highlighting the very real threat to HE without such action. Both types of practitioners are equally valued and respected in her discussion and drawing on theory, policy and experience this article forms a good opinion piece. It'll be interesting in particular to see what further study of the contribution and activity of learned societies in this area reveals.", "responses": [ { "c_id": "1881", "date": "23 Mar 2016", "name": "Anne Tierney", "role": "Author Response", "response": "The area of learned societies in one within which there is a lot of potential to support teaching and learning, and many learned societies include such in their conferences. We shall see over the coming years what the outcome of this is, as there is an appetite for support, which I believe will increase once the substance of TEF is known.Thank you for the review, and I look forward to any comments you have on this in the future." } ] }, { "id": "12752", "date": "22 Mar 2016", "name": "Joy Perkins", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis opinion piece is an extremely timely, topical and well-written article which highlights a range of important points regarding the two communities of practice (research-focused and teaching focused) which exist within universities and across the HE sector. There clearly needs to be a re-balance between the tensions of teaching and research and the author proposes approaches to help meet and unite the needs of both communities of practice through for example, an enhanced role for learned societies. To strengthen the article a paragraph recognising the role of the UK Professional Standards Framework (UKPSF) and the Higher Education Academy (HEA) Fellowship recognition scheme in raising the profile of teaching to all academics would be a valuable addition. HEA Fellowship status is a quality assurance indicator in UK universities regarding standards in teaching and supporting students’ learning and is often implemented as a KPI target in many institutions. Research experience is often relevant to an individual’s HEA Fellowship application if it relates to their university learning and teaching. Is this a further opportunity to extend the ‘brokering model’ proposed by the author? There is also potential to unite both communities of practice at HEA Writing Retreats and HEA recognition events, these CPD opportunities help support both communities to network and communicate their expertise.The article is acceptable for approval with a few minor corrections, it should be the Society for Experimental Biology (lower case t) – Introduction section, line 3 and the same applies to the Higher Education Academy (Introduction section, line 2).", "responses": [ { "c_id": "1880", "date": "23 Mar 2016", "name": "Anne Tierney", "role": "Author Response", "response": "The suggestion to include UKPSF and HEA fellowship is a sensible one, and I shall include a paragraph in the paper. Thank you very much for the suggestion, and thank you for taking the time to review the paper." } ] } ]	1	https://f1000research.com/articles/5-280
https://f1000research.com/articles/5-274/v1	03 Mar 16	{ "type": "Research Article", "title": "Chromas from chromatin: sonification of the epigenome", "authors": [ "Davide Cittaro", "Dejan Lazarevic", "Paolo Provero", "Dejan Lazarevic", "Paolo Provero" ], "abstract": "The epigenetic modifications are organized in patterns determining the functional properties of the underlying genome. Such patterns, typically measured by ChIP-seq assays of histone modifications, can be combined and translated into musical scores, summarizing multiple signals into a single waveform. As music is recognized as a universal way to convey meaningful information, we wanted to investigate properties of music obtained by sonification of ChIP-seq data. We show that the music produced by such quantitative signals is perceived by human listeners as more pleasant than that produced from randomized signals. Moreover, the waveform can be analyzed to predict phenotypic properties, such as differential gene expression.", "keywords": [ "Sonification", "Chromatin", "Music", "Information", "Epigenomics" ], "content": "Introduction\n\nSonification is the process of converting data into sound. Sonification itself has a long, yet punctuated, story of applications in molecular biology, several algorithms to translate DNA1 or protein sequences2,3 to musical scores have been proposed. The same principles have also been extended to the analysis of complex data4 showing that, all in all, sonification can be used to describe and classify data. This approach is sustained by the idea that music is acknowledged as a way to deliver information5. Indeed, the very same procedures may also be applied for recreational purposes.\n\nOne of the limitations of sonification of actual DNA and protein sequences is their intrinsic conservative nature. Assuming the differences in two individual genomes are, on average, one nucleotide every kilobase6, the corresponding musical scores would have little differences.\n\nOn the contrary, dynamic ranges typical of transcriptomic and epigenomic data may provide a richer source for sonification.\n\nIn this work we describe an approach to convert ChIP-seq signals, and in principle any quantitative genomic feature, into a musical score. We started working on our approach for amusement mainly, and we realized that the sonificated chromatin signals were surprisingly harmonious. We then tried to assess some properties of the music tracks we were able to generate. We show that the emerging sounds are not random and instead appear more melodious and tuneful than music generated from randomized notes. We also show that different ChIP-seq signals can be combined into a single musical track and that tracks representing different conditions can be compared allowing for the prediction of differentially expressed genes.\n\nExamples of sonification for various genomic loci are available at https://soundcloud.com/davide-cittaro/sets/k562.\n\n\nDefinitions\n\nMIDI: MIDI (Musical Instrument Digital Interface) is a standard that describes protocols for data exchange among a variety of digital musical instruments, computers and related devices. MIDI format encodes information about note notation, pitch, velocity and other parameters controlling note execution (e.g. volume and signals for synchronization).\n\nMIDI file format: a binary format representing MIDI data in a hierarchical set of objects. At the top of hierarchy there is a Pattern, which contains a list of Tracks. A Track is a list of MIDI events, encoding for note properties. MIDI events happen at specific time, which is always relative to the start of the track.\n\nMIDI Resolution: resolution sets the number of times the status byte is sent for a quarter note. The higher the resolution, the more natural the sound is perceived. Resolution is the number of Ticks per quarter note. At a specific resolution R, Tick duration in microseconds T is related to tempo (expressed in Beats per Minute, BPM) by the following equation\n\nT=60RBPM\n\n\nResults\n\nIn order to translate a single ChIP-seq signal track to music we bin the signal over a specified genomic interval (i.e. chrom:start-end) into fixed-size windows (e.g. 300 bp) and note duration will be proportional to the size of such windows. As we are dealing with MIDI standard, we let the user specify track resolution and the number of ticks per window (see Definitions); the combination of these parameters defines the duration of a single note. The default parameters associate a bin of 300 bp with one quaver (1/8 note).\n\nIn order to define the note pitch, we take the logarithm of the average intensity of the ChIP-seq signal in a genomic bin. The sounding range of the whole signal is discretized in a predefined number of semitones. At default parameters, the range is binned into 52 semitones, covering four octaves. In order to introduce pauses, the lowest bin of the signal range represents a rest. If two consecutive notes or rests fall in the same bin, we merge them in one note doubling its duration.\n\nUsing this approach, any ChIP-seq signal can be mapped to a chromatic scale. We implemented the possibility to map a signal on a different scale (major, minor, pentatonic…); to this end, intensity bin boundaries are merged according to the definition of a specific scale (Figure 1). MIDI tracks produced in this way can be then imported into a sequencer software where they can be further processed, setting tempo and time signature.\n\nChIP-seq values (H3K4me3 in the example) are binned in fixed-size intervals over the genome. Each interval corresponds to a 1/8 note. Average values of log-transform of read counts in each genomic bin (red lines) are matched into predefined number of semitones (chromatic scale). Notes may be mapped to a specified scale (major and minor scales are exemplified in the figure). Consecutive equal notes are merged in single note with double duration. Values falling in the first bin are considered rests.\n\nIn order to test whether sonification of chromatin marks are perceived as random patterns, we selected ten genomic regions and generated corresponding tracks based on the following histone modifications: H3K27me3, H3K27ac, H3K9ac, H3K36me3, H3K4me1, H3K4me2, H3K4me3, H3K9me3 (Supporting Audio files S1.1 to S10.1). For the same regions, we randomized genomic signal at base and bin level (Supporting Audio files S1.2 to S10.2). When data are randomized at base level, the average intensity is uniforms across the bins, resulting in a repeated note; this is largely expected as ChIP-seq signals are distributed on the genome according to a Poisson law7 or, more precisely, to a Negative Binomial law8.\n\nRandomization at bin level, instead, equals to shuffling notes during the execution. We administrated a questionnaire to a set of volunteers (n=8) not previously tested for education in music. Volunteers were asked to listen to each pair of original/random track and choose which track they felt was more appealing. Track order was randomized when testing different volunteers. Notably, in the majority of the cases (62/80) the music generated from genomic signal without randomization was judged more appealing. Results are significant to a Fisher-exact test (p=1.95e-3), suggesting that genomic signals contain information that can be recognized by human ear. The number of correct answers for each volunteer ranged from 5 to 10, with a median value of 8.\n\nOnce we assessed the existence of musical patterns in genomics signals, we were keen to explore if this kind of information could be exploited to identify biological features of samples. Since the epigenetic DNA modifications reflected by histone marks influence gene expression9, we tested if differences in musical tracks generated from various ChIP-seq signals reflects differences in gene expression of the corresponding loci. To this end, we downloaded ChIP-seq marks (H3K27me3, H3K27ac, H3K9ac, H3K36me3, H3K4me1, H3K4me2, H3K4me3, H3K9me3, Pol2b) and RNA-seq data for K562 and NHEK cell lines from the ENCODE project10. For each RefSeq locus we converted ChIP-seq signals to music with fixed parameters (see Methods). RNA-seq data were used to identify genes that are differentially expressed between the two cell lines, under a p-value <0.01 and \|logFC\|>1, according to recent SEQC recommendations11.\n\nA common way to classify music is based on summarization of track features after spectral analysis12,13. Such approach involves the summarization of track as Mel-Frequency Cepstral Coefficients (MFCC) that are subsequently clustered using Gaussian Mixture Models (GMM). A distance between tracks can then be defined as described in 14, who used it as a classifier for musical genres.\n\nWe tested if a similar approach could be used to develop a predictor of differential expression based on the distance between musical tracks generated from two cell lines.\n\nWe defined a distance between songs as described in methods and we optimized the parameters using as a training set the 250 genes with the most significant differential expression p-value and as many genes with the least significant p-value according to RNA-seq (Figure 2). We found that optimal performance is at MFCC=30 and GMM=10, with an AUC=0.609.\n\nEach square corresponds to a number of Mel-Frequency Cepstral Coefficients (MFCC) used to summarize signal and a number of centers for Gaussian Mixture Model (GMM). Colors are given by the corresponding Area Under the Curve (AUC).\n\nWe summarized tracks representing all RefSeq genes using such parameters, we then compared distances with differential expression performing a ROC analysis. Our results indicate that differences in information contained in musical representation of chromatin signals may be linked to differential expression, although power of prediction is limited (AUC=0.5184, p=1.4597e-03).\n\nAn additional issue we wanted to assess was if similarities between musical representation of chromatin status may be linked to the biology of the underlying genes. To this end, we calculated pairwise distances for all regions using parameters identified above on K562 cell line. Hierarchical clustering of the distance matrix identifies eight major clusters (Figure 3, left). We performed Gene Ontology Enrichment analysis on each cluster, here represented as word cloud of significant terms (Figure 3, center, Supplementary table 1); we found that different clusters are linked to genes showing different biological properties. For example, some clusters (6, 7 and 8) were linked to regulation of cell cycle, others were linked to metabolic processes (2 and 5) or vesicle transport (3 and 4). We also evaluated the distribution of expression (expressed as log(RPKM)) of the underlying genes (Figure 3, right); we found that regions clustered by the distance between musical tracks broadly reflects groups of genes with different level of expression, spotting clusters of higher expression (cluster 5) or lower expression (clusters 2 and 3); assessment of statistical significance of differences in distribution of gene expression values among clusters is presented in Table 1.\n\nEach cluster broadly corresponds to specific biological properties according to Gene Ontology enriched terms (middle). Level of expression of genes included in each cluster show specific distributions (right).\n\nTests showing significant difference (p ≤ 0.05) are presented in bold face.\n\n\nDiscussion\n\nChromatin shape and genome function are governed, among several factors, by the coordinated organization of epigenetic marks15. Modifications of such marks are dynamic and are fine-tuned during the life of a cell or an organism. Analysis of histone modifications, as well as transcription factors and other proteins binding DNA, by ChIP-seq already described patterns of enrichment that are specific to their relative function16,17. Analysis of combinatorial patterns of histone modifications already unveiled its potential in understanding functional properties of the genome18,19 and the cross-talk among multiple chromatin marks20.\n\nWe show, in this work, that the information carried by multiple histone modifications can be caught in a human-friendly way by translating ChIP-seq signals into musical scores. Although the investigation of the psychological factors that underlie tuneful perception of sonificated genomic signals is out of the scope of this manuscript, our results suggest that human hearing is able to perceive patterns conveying information encoded in ChIP-seq data analyzed and to distinguish from random noise.\n\nWe automated the analysis of differences between musical tracks using an established method based on summarization of spectral data. By this approach, we investigated the possible link between differences in ways chromatin sounds and phenotypic features. Our results suggest that differences in transcript levels can be predicted by the differences of sonificated genomic regions, although performances of such approach are limited. We reasoned that many factors may explain such poor results: first of all there is a vast space of parameters that can be tuned to create a single musical track and we still lack methods to explore it efficiently. In addition, the Mel scale used to summarize audio signal has been developed to match human capabilities to perceive sound21, hence it may not be optimal for the comparison of the tracks generated in this work.\n\nIt has already been shown that it is possible to predict levels of gene expression starting from chromatin states, although the method used to perform chromatin segmentation has a large impact on such predictions22. In this work we found that differences in chromatin-derived music reflects, to some extent, differences in the level of expression of underlying genes and their related biology.\n\nTo conclude, although we cannot advocate the usage of musical analysis as universal tool to analyze biological data yet, we confirm that quantitative features on the genome are patterned and contain information, hence can be converted into sounds that are perceived as musical. We limited our analysis on specific chromatin modifications, but in principle any quantitative genomic feature can be converted and integrated into a musical track. The choice of parameters and instruments has been standardized for the analysis presented, for illustrative purpose we show that different signals from the same region can be combined using different instruments (https://soundcloud.com/davide-cittaro/random-locus-blues) and signals from different genomic regions can be merged (https://soundcloud.com/davide-cittaro/non-homologous-end-joining).\n\n\nMaterials and methods\n\nRaw data for various modifications were downloaded from GEO database (GSE26320, http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE26320). Read tags were aligned to human genome (hg19) using BWA aligner v0.7.223. Alignments were converted to bigwig tracks24 after filtering for duplicates and quality score higher than 15:\n\n\n\nIn order to define regions to be converted to music scores, we selected intervals around RefSeq gene definition, from 1kb upstream of TSS to 2kb downstream of TES. ChIP-seq signals were firstly converted to MIDI using custom scripts (https://bitbucket.org/dawe/enconcert) according to parameters defined in Table 2. MIDI tracks belonging to the same region from the same sample were merged into a single MIDI file, converted to WAV format using timidity software v2.14.0 (http://timidity.sourceforge.net), with the exception of tracks presented as Supplementary audio files which have been processed with GarageBand software v10.1.0 (Apple Inc., Cupertino, USA).\n\nIn order to compare four samples for each converted genomic region, we extracted MFCC using python_speech_features library (https://github.com/jameslyons/python_speech_features). Selected components were then clustered using Gaussian Mixture Models, implemented in scikit-learn python library 0.15.2 (http://scikit-learn.org). Distances between two tracks were evaluated using Hausdorff distance (H) between GMM clusters. Briefly, we first calculate all pairwise distances between GMM clusters using Bhattacharyya distance (B) for multivariate normal distributions as\n\nB=18(μ0−μ1)TP−1(μ0−μ1)+12log⁡\|P\|\|𝛴0\|\|𝛴1\|\n\nwhere\n\nP=𝛴0+𝛴12\n\nthen, as GMM are not ordered, we take the Hausdorff distance (H) as the maximum between the row-wise and column-wise minimum of the pairwise distances between two GMM sets. ROC analysis on music distances was performed over the value of D, defined as\n\nD=log⁡(1+b¯)−log⁡(1+w¯)\n\nwhere\n\nw¯=H(K562a,K562b)+H(NHEKa,NHEKb)2\n\nis the average of distances between replicates and\n\nb¯=H(K562a,NHEKa)+H(K562a,NHEKb)+H(K562b,NHEKa)+H(K562b,NHEKb)4\n\nis the average of pairwise distances among different cell lines.\n\nRNA-seq tags were downloaded from GEO archive (GSE30567, http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE30567) and aligned to human reference genome (hg19) using STAR aligner v2.3.025. Read counts over RefSeq intervals were extracted using bedtools v.2.24.026. Discrete counts were normalized with TMM27, differential gene expression was evaluated using the voom function implemented in limma v.3.26.728 with a simple contrast between two cell lines. Genes were considered differentially expressed under a p-value lower than 0.01 and absolute logarithm Fold Change higher than 1.\n\nCluster analysis was performed on replicate 1 of K562 dataset. We calculated all pairwise Hausdorff distances among genomic loci as defined above. Data were clustered using the Ward method. Enrichment analysis was performed using online Enrichr suite29. Word clouds were created with world_cloud python package (https://github.com/amueller/word_cloud) using text description of ontologies having positive Enrichr combined score. Differential expression among clusters was evaluated using Mann-Whitney U-test.\n\n\nConsent\n\nWritten informed consent for publication was obtained from the study participants.\n\n\nData availability\n\nFigshare: Supplementary audio files for ‘Chromas from chromatin: sonification of the epigenome’, doi: 10.6084/m9.figshare.307954030\n\nFigshare: Supplementary table for ‘Chromas from chromatin: sonification of the epigenome’, doi: 10.6084/m9.figshare.307954331\n\n\nSoftware availability\n\nhttps://bitbucket.org/dawe/enconcert\n\nhttps://zenodo.org/record/4594332\n\nMIT License https://opensource.org/licenses/MIT", "appendix": "Author contributions\n\n\n\nDC, DL and PP designed the experiment and wrote the manuscript; DC performed the analysis.\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\nAuthors would like to acknowledge Piotr Traczyk (Soltan Institute for Nuclear Studies, Warsaw, Poland) who inspired our research with his sonification of the Higgs Boson. Authors would like to thank all collaborators and relatives who kindly sacrificed their time to listen to music generated while this work was developed.\n\n\nReferences\n\nOhno S, Ohno M: The all pervasive principle of repetitious recurrence governs not only coding sequence construction but also human endeavor in musical composition. Immunogenetics. 1986; 24(2): 71–78. PubMed Abstract \| Publisher Full Text\n\nKing RD, Angus CG: PM--protein music. Comput Appl Biosci. 1996; 12(3): 251–252. PubMed Abstract \| Publisher Full Text\n\nTakahashi R, Miller JH: Conversion of amino-acid sequence in proteins to classical music: search for auditory patterns. Genome Biol. 2007; 8(5): 405. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLarsen P, Gilbert J: Microbial bebop: creating music from complex dynamics in microbial ecology. PLoS One. 2013; 8(3): e58119. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJuslin PN: What does music express? Basic emotions and beyond. Front Psychol. 2013; 4: 596. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\n1000 Genomes Project Consortium, Abecasis GR, Auton A, et al.: An integrated map of genetic variation from 1,092 human genomes. Nature. 2012; 491(7422): 56–65. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang Y, Liu T, Meyer CA, et al.: Model-based analysis of ChIP-Seq (MACS). Genome Biol. 2008; 9(9): R137. 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\nKouzarides T: Chromatin modifications and their function. Cell. 2007; 128(4): 693–705. PubMed Abstract \| Publisher 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\nSEQC/MAQC-III Consortium: A comprehensive assessment of RNA-seq accuracy, reproducibility and information content by the Sequencing Quality Control Consortium. Nat Biotechnol. 2014; 32(9): 903–14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLogan B: Mel Frequency Cepstral Coefficients for Music Modeling. ISMIR. 2000. Reference Source\n\nDopler M, Schedl M, Pohle T, et al.: Accessing Music Collections Via Representative Cluster Prototypes in a Hierarchical Organization Scheme. ISMIR. 2008. Reference Source\n\nAucouturier JJ, Pachet F, Sandler M: “The way it Sounds”: timbre models for analysis and retrieval of music signals. IEEE Trans Multimedia. 2005; 7(6): 1028–1035. Publisher Full Text\n\nZhou VW, Goren A, Bernstein BE: Charting histone modifications and the functional organization of mammalian genomes. Nat Rev Genet. 2011; 12(1): 7–18. PubMed Abstract \| Publisher Full Text\n\nBarski A, Cuddapah S, Cui K, et al.: High-resolution profiling of histone methylations in the human genome. Cell. 2007; 129(4): 823–837. PubMed Abstract \| Publisher Full Text\n\nValouev A, Johnson DS, Sundquist A, et al.: Genome-wide analysis of transcription factor binding sites based on ChIP-Seq data. Nat Methods. 2008; 5(9): 829–834. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nErnst J, Kellis M: ChromHMM: automating chromatin-state discovery and characterization. Nat Methods. 2012; 9(3): 215–216. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSong J, Chen KC: Spectacle: fast chromatin state annotation using spectral learning. Genome Biol. 2015; 16(1): 33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPerner J, Lasserre J, Kinkley S, et al.: Inference of interactions between chromatin modifiers and histone modifications: from ChIP-Seq data to chromatin-signaling. Nucleic Acids Res. 2014; 42(22): 13689–13695. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStevens SS, Volkmann J, Newman EB: A Scale for the Measurement of the Psychological Magnitude Pitch. J Acoust Soc Am. 1937; 8(3): 185–190. Publisher Full Text\n\nHoffman MM, Ernst J, Wilder SP, et al.: Integrative annotation of chromatin elements from ENCODE data. Nucleic Acids Res. 2013; 41(2): 827–841. 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): 589–595. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKent WJ, Zweig AS, Barber G, et al.: BigWig and BigBed: enabling browsing of large distributed datasets. Bioinformatics. 2010; 26(17): 2204–7. 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\nQuinlan AR: BEDTools: The Swiss-Army Tool for Genome Feature Analysis. Curr Protoc Bioinformatics. 2014; 47: 11.12.1–11.12.34. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRobinson MD, Oshlack A: A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol. 2010; 11(3): R25. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRitchie ME, Phipson B, Wu D, et al.: limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015; 43(7): e47. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen EY, Tan CM, Kou Y, et al.: Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool. BMC Bioinformatics. 2013; 14: 128. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCittaro D, Lazarevic D, Provero P: Supplementary audio files for ‘Chromas from chromatin: sonification of the epigenome’. Figshare. 2016. Data Source\n\nCittaro D, Lazarevic D, Provero P: Supplementary table for ‘Chromas from chromatin: sonification of the epigenome’. Figshare. 2016. Data Source\n\nCittaro D: Enconcert. Zenodo. 2016. Data Source" }	[ { "id": "12733", "date": "15 Mar 2016", "name": "Tao Liu", "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\nTo translate functional genomics data in ChIP-seq to music pieces is entertaining. Cittaro et al's work showed that, the chromatin data which has wider dynamic range is suitable (in my opinion perhaps more suitable than DNA/protein sequences in spite of lacking supporting evidence) for making appealing music. I feel excited while reading the manuscript and listening to the music pieces made by the authors at the same time.This work definitely has the novelty and importance especially for education and popular science. I believe more students and general public will be attracted by the chromatin songs then start to learn the science behind them. Because I don't have the expertise of music theory, I won't comment on the quality of the chromatin music or the methods to tune the music. Since authors showed the music patterns may also reflect the biology on the chromatin and may be associated with gene expression. I will focus on these. Authors translated chromatin marks ChIP-Seq data from human K562 and NHEK cell lines at refSeq gene bodies from upstream 1k of TSS to 2kb downstream of TES, then tried to study if the music patterns can match the differential gene expression inferred from RNA-Seq. The prediction power was quite low, as pointed out by the authors, with an AUC of 0.52 ( just a little better than random) with the optimal combination of parameters got from a subset of genes. A natural question is that how well the prediction is by directly using chromatin signals, such as the tags pileup. By comparing with this, we will see whether the underlying biology has been kept or lost during the approach. Authors showed that the similarities, in terms of hierarchical clustering, of musical representation at gene bodies, from the K562 cell line, can be linked to gene functions and similar gene expression. However I found it's hard to see the consistency of the similarities of music with the similarities of gene function annotations. For example, the cluster 7 and 8 are the closet pair of clusters, although authors claimed that clusters 6,7,8 were linked to regulation of cell cycle, I can't see such words from the word clouds of the middle panel of figure 3. Instead, I can see that the cluster 1, 3, 5 and 7 all have the same term of biological function 'protein' although they are apart according to the hierarchical clustering. The gene expression analysis is also confusing while looking at the right panel of figure 3 and table 1. It seems that for all the clusters there are two major distributions of gene expression levels from the violin plots -- a big one at log(RPKM) less than 1 and a small one around log(RPKM) of 2. Does that mean the big population of each cluster are just random noises of weakly expressed genes (logRPKM<1)? The numbers of genes in each cluster were also missing in the manuscript. I wonder perhaps the cluster 2 contains very few genes since although visually it seems the distribution of gene expression of cluster 2 is quite different with all the other clusters, the p-values of the row 2 of table 1 (cluster 2 against 3, 4, 5, 6,7 and 8) are all very small, indicating no significant difference. Additionally, I should bring my comment of point 1 here as well. How the clustering works while checking only the raw ChIP-Seq pileup? Comments on method section:For the equations of 'comparison of WAV track', the notations were not explained clearly. Description on how the RPKM was calculated is missing. The bases of logarithm functions are missing for the 'log(RPKM)' of gene expression level and the 'absolute logarithm fold change' used to define differential expressed genes.", "responses": [] }, { "id": "12735", "date": "18 Mar 2016", "name": "Ho-Ryun Chung", "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“Chromas from chromatin: sonification of the epigenome” by Cittaro et al. deals with the transformation of ChiP-seq profiles into music. The authors transform BigWig files into notes by discretizing the logarithm of the signal intensity. Test individuals perceive the resulting music as more appealing than randomized controls. The authors explore the possibility to use the music to discern differentially expressed genes in two cell lines from unchanged ones. The classification accuracy is a little bit better than random guessing. They also use the similarity of the music to cluster genes. They claim that the clustering reveals genes with similar biological properties. They also show that the clustering is correlated to the gene expression level. I find the idea really interesting. It may help the vision impaired to gather information about ChIP-seq tracks or other quantitative vector like information. It may be used to train individuals to recognize certain epigenomic features, like promoters and enhancers. I find it not so surprising that the chromatin music is more appealing to test persons than randomized controls, because of the smoothness of the ChIP-seq profile data. Smoothness guaranties that subsequent notes are close by forming a more melodious line. Once randomized it results in random notes, which may be far apart and are perceived by most as not so appealing. Thus, humans can distinguish between smooth signals and signals that change abruptly and perceive the former more appealing than the latter. But clearly there is information in the music that can be exploited by the human ear. The analyses about differential gene expression and gene clustering use the music as a feature in computer-aided classification tasks. These analyses show that there is some information in the music that a computer can recognize and use for classification. However, a base line using just the ChIP-seq profiles without turning them to music is missing. Without such a base line it remains hard to judge whether the reported results are meaningful or not. Moreover, I think that the classification results shown in Figure 3 are likely to reflect gene expression differences rather than gene function clustering. Are the p-values reported in Table 1 for a one- or two-sided test? I do not understand why cluster 2 is not different from all the others except cluster 1. I think the real potential in chromatin music is not so much in its use in machine learning approaches – it is much better suited for humans learning chromatin states and the like. Humans may be able to recognize patterns that a machine cannot. It would be really interesting to compare a human made chromatin segmentations using chromatin music with a segmentation generated from the ChIP-seq signals by a computer. Finally, the proposed tool can be used to reach out to the public and demonstrate ideas about epigenomic states etc. using an easily accessible medium such as music.", "responses": [] }, { "id": "12734", "date": "22 Mar 2016", "name": "Federico M Giorgi", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article \"Chromas from chromatin: sonification of the epigenome\" proposes a new and original method to sonify (i.e. convert into music) data information coming from the analysis of the epigenome.The paper is extremely well written and it should be indexed on the basis of being the first to try this kind of review. The results of Cittaro et al.'s work, although without an immediate applicability in the field of biomedicine, is by itself a small scientifical breakthrough.My only concerns regard the applicability of the sonification method as a truly alternative way to detect biological properties.The authors imply that similar biological features (e.g. belonging to a particular pathway, differential expression) could be discerned by listening to gene-centered audio tracks. I listened to such tracks and I concur, but to be scientifically complete, the authors may think (even as a follow up paper) to test this on a wider subject set. I would propose a psychological study with a blind panel of human subjects, who are then asked, after listening to some biological properties on a \"training set\", to find genes with similar properties on a \"test set\".", "responses": [] } ]	1	https://f1000research.com/articles/5-274
https://f1000research.com/articles/4-1145/v1	27 Oct 15	{ "type": "Observation Article", "title": "A paradoxical synergism between Resveratrol and copper (II) with respect to degradation of DNA and RNA", "authors": [ "Siddharth Subramaniam", "Iqbal Vohra", "Aishwarya Iyer", "Naveen K Nair", "Indraneel Mittra", "Siddharth Subramaniam", "Iqbal Vohra", "Aishwarya Iyer", "Naveen K Nair" ], "abstract": "Resveratrol (R), a plant polyphenol, is known to reduce Cu (II) to Cu (I) generating reactive oxygen species that can cleave plasmid DNA. Here we report a surprising observation of a paradoxical synergistic effect between R and Cu whereby plasmid DNA cleaving / degrading activity of R-Cu increased progressively as the ratio of R to Cu was increased i.e., the concentration of Cu was successively reduced with respect to a fixed concentration R. Whereas cleavage of plasmid DNA occurred at low molar ratios of R to Cu, at higher ratios, complete degradation of DNA was achieved. By further increasing the ratio, whereby the concentration of Cu was reduced to very low levels, the DNA degrading activity of R-Cu was lost. This paradoxical synergistic effect is also seen with respect to eukaryotic genomic DNA and RNA. Since R-Cu may have anti-cancer and anti-viral activities, our findings may not only help to improve the therapeutic efficacy of R-Cu but also reduce its toxic side effects with the use of low concentration of Cu.", "keywords": [ "Resveratrol", "copper", "pro-oxidant activity", "plasmid DNA cleavage", "plasmid DNA degradation", "eukaryotic DNA degradation", "RNA degradation" ], "content": "Introduction\n\nResveratrol (R) is a poly-phenolic stilbenoid naturally present in the skin of red grapes and other fruits and berries, peanuts and also in the roots of Japanese knotweed1. R has been shown to have multiple health benefits that include life extension, cancer prevention, cardio-protection, neuro-protection and anti-diabetic, anti-inflammatory and anti-viral activities2–8. These actions are thought to be mediated through its intrinsic anti-oxidant properties and the ability of R to activate SIRT19–11. However most of the positive effects exhibited by R could not be replicated in clinical trials possibly because of its low bio-availability12,13.\n\nCopper (Cu) is an essential micronutrient, and because of its role as a metal co-factor, has the ability to generate reactive oxygen species (ROS), viz., O2-. and •HO radicals14. Fukuhara and Miyata were first to show that R can act as a pro-oxidant in the presence of Cu and cause oxidative DNA cleavage in a pBR322 plasmid assay15. R forms a complex with Cu (II), leading to its reduction to Cu (I) with concomitant production of ROS which is responsible for DNA scission16. Resveratrol-copper (R-Cu) was shown to be active in biological systems as evidenced by its ability to inactivate bacteriophages8 and to cause fragmentation of DNA of human lymphocytes in vitro17. These findings have led to the proposal that R-Cu could be used in the prevention and treatment of cancer17,18.\n\nThe above studies have used variable molar ratios of R:Cu which have usually been of the order of 1:1 to 2:1. Here we report a surprising observation that DNA and RNA cleaving and/or degrading activity of R-Cu increases as the ratio of R to Cu is sequentially increased (i.e., the concentration of Cu is sequentially decreased with respect to a fixed concentration of R). The activity was lost when the Cu concentration was reduced to very low levels.\n\n\nMethods\n\nIsolation of plasmid pTRIPZ DNA. Isolation of plasmid pTRIPZ DNA was performed using HiPurA plasmid DNA miniprep purification spin kit (Hi-Media) as per manufacturer’s instructions. Briefly, the transformed bacterial culture (Escherichia coli DH5α containing plasmid pTRIPZ. Invitrogen, USA) was harvested, lysed and centrifuged. The pellet obtained was applied to a silica column and high salt (3M Potassium acetate, pH 5.5) was used to allow binding of plasmid DNA to the silica column. Washing for removal of contaminants was followed by elution of plasmid DNA in DNA binding buffer.\n\nEukaryotic genomic DNA. Jurkat (human lymphoblastic leukemia) cells were used for isolation of genomic DNA. Cells were procured from American Type Culture Collection and were grown in RPMI 1640 (GIBCO By Life technologies Cat No.23400-21) with 10% FBS (GIBCO By life technologies Cat No.26140-079). The Wizard® Genomic DNA purification kit (Promega) was employed for isolation of DNA. Jurkat cells (2 × 106) were harvested and given three PBS washes followed by treatment with nuclei lysis solution. Genomic DNA was isolated as per manufacturer’s protocol.\n\nIsolation of eukaryotic RNA. Jurkat cells at the exponential phase of growth (approximately 5 × 106) were washed thrice in PBS and RNA was isolated using Trizol® reagent (Life Technologies, Carlsbad, CA, USA) as per manufacturer’s protocol.\n\nPlasmid pTRIPZ DNA (500ng), eukaryotic genomic DNA (500ng) and eukaryotic RNA (2μg) were suspended in Tris-EDTA buffer. R-Cu was dissolved in 20 μl of following solvents: 50% ethanol; 50% acetonitrile; 3mM NaOH or distilled water in varying molar ratios of R to Cu as indicated in the text and incubated with plasmid or genomic DNA and eukaryotic RNA at 37°C for 1hr. After incubation, the different DNA samples were electrophoresed on a 1% agarose gel using a horizontal electrophoresis unit (Hoefer) at a constant voltage of 100V. In case of eukaryotic RNA, the mixtures after incubation were subjected to 0.8% agarose gel electrophoresis at 75 volts for 90 minutes. The gel-documentation system, EC-3 Imaging system from UVP (Ultra Violet Products, USA) was used to record the images.\n\n\nObservation\n\nWe observed that when we increased the ratio of R to Cu (by reducing the concentration of Cu with respect to a fixed concentration of R) there was an enhancement of cleavage/degradation of plasmid DNA (Figure 1–Figure 4). This phenomenon was dependent on the starting concentration of R-Cu. For example, cleavage of supercoiled plasmid DNA was observed at a starting concentration of 100μM at molar ratios of 1:1 and 1:0.2 (lanes 5 and 6; Figure 1). However, with successive increases in starting concentration of R-Cu to 500μM, 1mM and 5mM, DNA cleaving activity was progressively enhanced such that complete cleavage was achieved at successively higher ratios of R to Cu (i.e., with decreasing Cu concentration) (Figure 2–Figure 4). At high starting concentrations viz., 1mM and 5mM, degradation rather than cleavage of DNA was observed. These data indicated that the DNA cleaving/degrading activity of R-Cu increases as the ratio of R to Cu is successively increased thereby suggesting the existence of a paradoxical synergistic relationship between R and Cu with respect to DNA cleavage/degradation. The data also show that the extent of cleavage/degradation is positively correlated with the starting concentrations of R and Cu. Figure 5 and Figure 6, in which genomic DNA and RNA respectively were used (starting molar ratio of R to Cu of 5mM:5mM), a similar synergistic pattern was observed.\n\nStarting concentration R 100µM : Cu 100µM. Reaction performed in 50% ethanol.\n\nStarting concentration R 500µM : Cu 500µM. Reaction performed in 50% ethanol.\n\nStarting concentration R 1mM : Cu 1mM. Reaction performed in 50% ethanol.\n\nStarting concentration R 5mM : Cu 5mM. Reaction performed in 50% ethanol.\n\nStarting concentration R 5mM:Cu 5mM. The reactions were performed in 50% ethanol.\n\nStarting concentration R 5mM:Cu 5mM. Reactions were performed in 50% ethanol.\n\nThe above experiments were done in 50% ethanol (Figure 1–Figure 4). A similar synergism was also observed when experiments were done using other solvents, namely, 50% acetonitrile (Figure 7), 3mM NaOH (Figure 8) and water (Figure 9) confirming the robust nature of this synergistic phenomenon.\n\nReactions were performed in 50% Acetonitrile.\n\nReactions were performed in 3mM NaOH.\n\nReactions were performed in water.\n\n\nDiscussion\n\nSpectroscopic studies using an analogue of Resveratrol, namely Piceatannol (3,3',4,5'-tetrahydroxy-trans-stilbene; Pice), have shown that Pice-Cu(II) induced DNA cleavage involves the Haber Weiss and Fenton reactions19. DNA cleavage is a result of hydroxyl radical formation and the Cu (II) to Cu (I) redox cycle generated ROS production19. Our experiments using R-Cu reported here suggest that the Cu (II) – mediated oxidation of R is in a catalytic mode via Cu (II) – Cu (I) redox cycling; Cu (II) acts as a catalyst with an optimum dosage depending on the starting concentration of R-Cu. However, our unexpected finding of increasing DNA and RNA cleavage/degradation with decreasing concentration of Cu remains currently unexplained and requires further investigation.\n\nSince R-Cu may have anti-cancer and anti-viral activities8,18, our finding may not only help to improve the therapeutic efficacy of R-Cu but also reduce its toxic side effects with the use of low concentration of Cu.", "appendix": "Author contributions\n\n\n\nSS, IV and AI performed the experiments and interpreted the results. NKN wrote the manuscript. IM designed the experiments, interpreted the results and wrote the manuscript. All authors have seen and agreed to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study was supported by the Department of Atomic Energy, Government of India, through its grant CTCTMC to Tata Memorial Centre awarded to IM.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nBurns J, Yokota T, Ashihara H, et al.: Plant foods and herbal sources of resveratrol. J Agric Food Chem. 2002; 50(11): 3337–3340. PubMed Abstract \| Publisher Full Text\n\nHector KL, Lagisz M, Nakagawa S: The effect of resveratrol on longevity across species: a meta-analysis. Biol Lett. 2012; 8(5): 790–793. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSuh DH, Kim Mi-K, Kim HS, et al.: Cancer-specific Therapeutic Potential of Resveratrol: Metabolic Approach against Hallmarks of Cancer. J FFHD. 2013; 3(8): 332–343. Reference Source\n\nPollack RM, Crandall JP: Resveratrol: therapeutic potential for improving cardiometabolic health. Am J Hypertens. 2013; 26(11): 1260–1268. PubMed Abstract \| Publisher Full Text\n\nMenard C, Bastianetto S, Quirion R: Neuroprotective effects of resveratrol and epigallocatechin gallate polyphenols are mediated by the activation of protein kinase C gamma. Front Cell Neurosci. 2013; 7: 281. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBhatt JK, Thomas S, Nanjan MJ: Resveratrol supplementation improves glycemic control in type 2 diabetes mellitus. Nutr Res. 2012; 32(7): 537–541. PubMed Abstract \| Publisher Full Text\n\nDas S, Das DK: Anti-inflammatory responses of resveratrol. Inflamm Allergy Drug Targets. 2007; 6(3): 168–173. PubMed Abstract \| Publisher Full Text\n\nAhmad A, Farhan Asad S, Singh S, et al.: DNA breakage by resveratrol and Cu(II): reaction mechanism and bacteriophage inactivation. Cancer Lett. 2000; 154(1): 29–37. PubMed Abstract \| Publisher Full Text\n\nLeonard SS, Xia C, Jiang BH, et al.: Resveratrol scavenges reactive oxygen species and effects radical-induced cellular responses. Biochem Biophys Res Commun. 2003; 309(4): 1017–1026. PubMed Abstract \| Publisher Full Text\n\nGusman J, Malonne H, Atassi G: A reappraisal of the potential chemopreventive and chemotherapeutic properties of resveratrol. Carcinogenesis. 2001; 22(8): 1111–1117. PubMed Abstract \| Publisher Full Text\n\nLagouge M, Argmann C, Gerhart-Hines Z, et al.: Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRT1 and PGC-11alpha. Cell. 2006; 127(6): 1109–1122. PubMed Abstract \| Publisher Full Text\n\nPonzo V, Soldati L, Bo S: Resveratrol: a supplementation for men or for mice? J Transl Med. 2014; 12: 158. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWalle T, Hsieh F, DeLegge MH, et al.: High absorption but very low bioavailability of oral resveratrol in humans. Drug Metab Dispos. 2004; 32(12): 1377–1382. PubMed Abstract \| Publisher Full Text\n\nLi Y, Kuppusamy P, Zweier JL, et al.: ESR evidence for the generation of reactive oxygen species from the copper-mediated oxidation of the benzene metabolite, hydroquinone: role in DNA damage. Chem Biol Interact. 1995; 94(2): 101–120. PubMed Abstract \| Publisher Full Text\n\nFukuhara K, Miyata N: Resveratrol as a new type of DNA-cleaving agent. Bioorg Med Chem Lett. 1998; 8(22): 3187–3192. PubMed Abstract \| Publisher Full Text\n\nFukuhara K, Nagakawa M, Nakanishi I, et al.: Structural basis for DNA-cleaving activity of resveratrol in the presence of Cu(II). Bioorg Med Chem. 2006; 14(5): 1437–1443. PubMed Abstract \| Publisher Full Text\n\nAzmi AS, Bhat SH, Hadi SM: Resveratrol-Cu(II) induced DNA breakage in human peripheral lymphocytes: implications for anticancer properties. FEBS Lett. 2005; 579(14): 3131–3135. PubMed Abstract \| Publisher Full Text\n\nHadi SM, Ullah MF, Azmi AS, et al.: Resveratrol mobilizes endogenous copper in human peripheral lymphocytes leading to oxidative DNA breakage: a putative mechanism for chemoprevention of cancer. Pharm Res. 2010; 27(6): 979–988. PubMed Abstract \| Publisher Full Text\n\nLi Z, Yang X, Dong S, et al.: DNA breakage induced by piceatannol and copper (II): Mechanism and anticancer properties. Oncol Lett. 2012; 3(5): 1087–1094. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "11205", "date": "12 Nov 2015", "name": "Renata Cozzi", "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 topic of this paper is very important and has interesting implications for the purpose of using resveratrol as chemopreventive agent. In fact the authors rightly refer to the paper by Azmi et al. where the problem of the potential breaking activity of resveratrol in human lymphocytes is addressed.However in my opinion the interpretation of the results of this manuscript is not always supported by the data presented.Referring to figures from 1 to 4 the authors state: “with successive increases in starting concentration of R-Cu to 500μM, 1mM and 5mM, DNA cleaving activity was progressively enhanced such that complete cleavage was achieved at successively higher ratios of R to Cu”. This is partially true in the sense that starting from the ratio R-Cu 1:1 the cleavage of supercoiled increases until 1:0.01 with the highest dose of resveratrol (5mM). At lower ratios no cleavage/degradation is observed. In other word it seems that the cleaving activity is due to resveratrol concentration increase rather than the ratio R-Cu.In fact the authors confirm this reading of the results (“The data also show that the extent of cleavage/degradation is positively correlated with the starting concentrations of R and Cu”) but suggest also “the existence of a paradoxical synergistic relationship” (even in the title) that is not at all supported by the data. The word “synergism” does not seem appropriate.As far as eukaryotic genomic DNA a similar behavior is showed: the cleavage/degradation of DNA is evident from ratio 1:0.2 to 1:0.002,not at lower ratios.I suggest:To use (certainly in the title and throughout the text) a less demanding word than “synergism”. Furthermore a synergistic effect would require also a statistical evaluation. To provide an explanation and/or an interpretative hypothesis about the showed effect present only in the middle ratios. To provide a more accurate description of figure 7, 8 and specially 9.", "responses": [ { "c_id": "1766", "date": "02 Mar 2016", "name": "Indraneel Mittra", "role": "Author Response", "response": "As suggested by the Reviewer, we have replaced the word \"synergism\" with \"relationship\" in the title and throughout the paper. As mentioned in the 'discussion' section, we are unable to provide any explanation for this paradoxical relationship between Resveratrol and copper. This is the reason why we have submitted the manuscript under \"Observation\" section of the Journal which stipulates \"We welcome Observation Articles describing a novel observation that may be unexpected, and possibly currently without explanation\". We would be delighted if the Referee could provide us some clues. We have now provided an accurate descriptions of Figures 7, 8 and 9.In order for the reader to easily grasp our findings depicted in multiple gel pictures, we have provided a 'summary table' of all the gel figures in a tabular format (Table) which reflects the paradoxical relationship between R and Cu more clearly. We trust that we have satisfactorily responded to all the suggestions of the referee and hope that she would now find the paper worthy of approval." } ] } ]	1	https://f1000research.com/articles/4-1145
https://f1000research.com/articles/4-103/v1	01 May 15	{ "type": "Research Article", "title": "The unfolded protein response and its potential role in Huntington ́s disease elucidated by a systems biology approach", "authors": [ "Ravi Kiran Reddy Kalathur", "Joaquin Giner-Lamia", "Susana Machado", "Kameshwar R S Ayasolla", "Matthias E. Futschik", "Ravi Kiran Reddy Kalathur", "Joaquin Giner-Lamia", "Susana Machado", "Kameshwar R S Ayasolla" ], "abstract": "Huntington ́s disease (HD) is a progressive, neurodegenerative disease with a fatal outcome. Although the disease-causing gene (huntingtin) has been known for over 20 years, the exact mechanisms leading to neuronal cell death are still controversial. One potential mechanism contributing to the massive loss of neurons observed in the brain of HD patients could be the unfolded protein response (UPR) activated by accumulation of misfolded proteins in the endoplasmic reticulum (ER). As an adaptive response to counter-balance accumulation of un- or misfolded proteins, the UPR upregulates transcription of chaperones, temporarily attenuates new translation, and activates protein degradation via the proteasome. However, persistent ER stress and an activated UPR can also cause apoptotic cell death. Although different studies have indicated a role for the UPR in HD, the evidence remains inconclusive. Here, we present extensive bioinformatic analyses that revealed UPR activation in different experimental HD models based on transcriptomic data. Accordingly, we have identified 58 genes, including RAB5A, HMGB1, CTNNB1, DNM1, TUBB, TSG101, EEF2, DYNC1H1 and SLC12A5 that provide a potential link between UPR and HD. To further elucidate the potential role of UPR as a disease-relevant process, we examined its connection to apoptosis based on molecular interaction data, and identified a set of 40 genes including ADD1, HSP90B1, IKBKB, IKBKG, RPS3A and LMNB1, which seem to be at the crossroads between these two important cellular processes.", "keywords": [ "Unfolded protein response (UPR)", "Huntington's disease", "Apoptosis", "UPR interactome", "HTT interactome" ], "content": "Introduction\n\nHuntington’s disease (HD) is an autosomal-dominant neurodegenerative disorder. Its symptoms include loss of motor control, cognitive decline, and behavioural abnormalities. In most cases, the onset of the disease occurs between the age of 35 and 50. The outcome is always fatal with a life expectancy following the disease onset of around 20 years.\n\nThe treatment of HD has remained symptomatic, as currently there is still no cure. The cause of HD is a mutation in a single gene called huntingtin (HTT). In HD patients, an expansion of the CAG repeat in exon 1 of huntingtin has been identified1. This mutation results in an extended stretch of polyglutamine close to the N-terminus of the Huntingtin protein (HTT), which is involved in multiple molecular functions2,3. Although the molecular cause has been known now for almost 20 years, the exact mechanisms leading to the observed massive cell death of neurons in the caudate nucleus of HD patients still await full clarification. A variety of processes such as excitotoxicity4,5, protein aggregation6–9 and transcriptional dysregulation10 have been suggested to contribute to neurodegeneration in HD. More recently, several studies have indicated that the unfolded protein response (UPR) might be implicated in neurodegenerative diseases including HD11–13.\n\nThe endoplasmic reticulum (ER) is a crucial organelle for the correct folding and modification of numerous proteins. Upon the accumulation of unfolded or misfolded protein in the ER, several transcriptional and translational mechanisms are triggered to ensure fidelity of protein folding14,15. This stress response is better known as the UPR. In particular, the UPR stress sensors Inositol-requiring protein-1 (IRE1), activating transcription factor 6 (ATF-6) and PKR-like ER kinase (PERK) are activated in mammalian cells when the ER exceeds its capacity for correct folding. As an adaptive response to counter-balance accumulation of un- or misfolded proteins, the UPR (i) upregulates transcription of chaperones, (ii) temporarily attenuates new translation, and (iii) activates protein degradation via the proteasome16. The main function of the UPR is re-establishing homeostasis by increasing the overall folding capacity. Although the primary role of UPR is an adaptive one, persistent ER stress can mediate toxicity and eventually lead to apoptosis through activation of JNK, ASK1 and caspase-12. Figure 1 depicts the different mechanisms and outcomes of UPR activation.\n\nOne explanation for how UPR sensors IRE1, PERK and ATF6 are activated is through detachment of chaperone BiP in the presence of excess un- or misfolded protein. This leads subsequently to the execution of a series of molecular processes with different effector functions86. As an adaptive response, the UPR up-regulates transcription of chaperones, temporarily attenuates new translation, and activates protein degradation via the proteasome. Persistent levels of ER stress, however, may trigger inflammatory pathways as an alarm signal in addition to caspase activation, leading ultimately to the induction of apoptosis. Mutant Huntingtin (mHTT) might provoke ER stress through interference with different processes such as vesicular transport or ER-associated degradation (ERAD) resulting in accumulation of (misfolded) protein in the ER.\n\nER stress and UPR have been indicated for a variety of neurodegenerative disorders, where protein misfolding plays a significant role17–19. For HD, finding a direct connection appears to be an enigma at first glance, since HTT is not located in the ER and thus its misfolding should not trigger UPR. However, several proposals have been put forward to describe how mutant HTT (mHTT) can induce an ER stress response12. For instance, experimental evidence from HD cell models suggests that cytosolic mHTT fragments strongly impair ER-associated protein degradation (ERAD), since mHTT entraps ERAD proteins20. This impairs the proper protein catabolism, causing a potential accumulation of misfolded proteins in the ER, effectively interfering with its correct functioning. Another alternative route towards ER stress in HD could be the perturbation of vesicular trafficking by mHTT resulting in a general protein overload in the ER or the disturbance of ER calcium homeostasis leading to a decreased folding capacity12.\n\nAlthough various lines of investigations have shown a potential role for UPR in the pathogenesis of HD, it remains difficult to assess its overall influence, given that the animal models and cell lines used in each individual study display great variability and distinct characteristics. Furthermore, most studies addressing the connection between UPR and HD focus on a small set of genes and proteins21,22. As the UPR presents a potentially important process in HD progression and a novel therapeutic target, we aimed to complement these previous studies with systematic and comprehensive bioinformatic analyses. Accordingly, using a systems biology approach, we gathered all available data and focused on detecting the activation of UPR during HD, and also elucidating the potential connection between UPR and apoptosis in HD.\n\nFirst, we assembled different sets of genes associated with the UPR and examined whether the included genes show differential expression in HD models or patients, when compared to controls. Next, we examined the promoter regions of upregulated UPR genes and detected significant enrichment of characteristic stress response elements. Additionally, we performed functional enrichment analysis on differentially expressed genes and found major biological processes implicated in UPR to be significantly over-represented. Furthermore, we assembled the UPR interactome and identified common proteins involved with apoptotic processes as well as with known HTT interactors, since those could provide crucial links between apoptosis and HD.\n\n\nMaterials and methods\n\nSince the UPR is a complex process, it is challenging to define a unique set of associated genes. Accordingly, we compiled three alternative gene sets that are either directly or indirectly involved in UPR, gathered from three distinct sources. The first termed, UPR-KEGG-GO (n=265), was derived from Gene Ontology23 (http://geneontology.org/page/go-database) (RRID:nif-0000-20935) and Pathway24,25 (http://www.genome.jp/kegg/pathway.html) (RRID:nlx_31015) databases indicated in Table 1. The second, referred to as UPR-interaction network (n=281), was generated by assembling molecular interactions of UPR components ATF6, ATF4, DDIT3, EIFAK3, ERNI and XBP1 using UniHI26 (http://www.unihi.org/) (RRID:nif-0000-03609) and HDNetDB databases (http://hdnetdb.sysbiolab.eu). The third gene set, labelled UPR-literature (n=2048) was compiled from published experimental results27–30 performed in yeast and human cells using high-throughput techniques such as yeast two-hybrid, microarrays and ribosome profiling coupled with next generation sequencing, as well as from text-mining of the GeneCards database31 (http://www.genecards.org) (RRID:nif-0000-02879) (Table 1).\n\nIn order to examine the genes involved in the cross-talk between UPR and apoptosis, we derived a list of genes that are either directly or indirectly involved in apoptosis from several different sources, namely the Gene Ontology database (GO:0006915; Apoptotic process; n=431), KEGG pathway (hsa04210; Apoptosis; n=88), Reactome pathway database (REACT_578.8; Apoptosis; n=148; http://www.reactome.org/) (RRID:nif-0000-03390) and literature reviews (n=85)32–36. All genes included were annotated to be involved in the induction of apoptosis, anti-apoptosis, regulation of apoptosis or were caspases (including both activators and inhibitors).\n\nFor establishing putative links to HD, we additionally put together two other gene sets:\n\n(i) HD therapeutic targets (HDTT) comprising 1033 genes. This set includes genes which were annotated by the curators of the HD Research CrossRoads database as being associated with HD based on experimental evidence, making them potential therapeutic targets. A detailed description of this gene set is provided elsewhere by Kalathur et al.37. The list of HDTT can be accessed at http://hdtt.sysbiolab.eu/.\n\n(ii) HTT interactors (HTT-int) including 1015 genes whose proteins have been shown to interact, or to be physically associated with HTT based on a diverse range of experiments. The large number of interactors can be explained through the inclusion of high-throughput affinity purification experiments, which frequently results in the addition of indirect interactions (e.g. within complexes). This set of interactors was obtained from the HDNetDB database (http://hdnetdb.sysbiolab.eu).\n\nAll HD gene expression data used for this study were downloaded from the Gene Expression Ominibus (GEO) database38 (http://www.ncbi.nlm.nih.gov/geo/) (RRID:nif-0000-00142). These data include gene expression from human brain and blood samples, human iPSCs, mouse, rat and yeast HD models, as well as murine cell lines (Table 2). All expression data sets were pre-processed using RMA (Robust Multi-array Average) implemented in R (available at http://www.r-project.org/) (RRID:nif-0000-10474) and analysed using several Bioconductor packages39,40 (RRID:nif-0000-10445). To enable the comparison across organisms, we mapped genes from mouse, rat, worm and yeast to orthologous human genes using HGNC Comparison of Orthology Predictions (HCOP) search tool (available online at http://www.genenames.org/cgi-bin/hcop), which is based on integrated data from HUGO Gene Nomenclature Committee (HGNC)41 (RRID:nif-0000-02955).\n\nWe performed gene set enrichment analysis (GSEA)42 (RRID:nif-0000-30629) comparing HD-associated expression to wild type or control data to identify differentially expressed genes. As input, we used the above-mentioned UPR gene sets and HD gene expression data. UPR genes were identified as significant when the enrichment score (ES) corresponded to a false discovery rate (fdr) ≤ 0.05 in HD gene expression data sets. For further analysis, we used only the genes present in the ‘UPR core enrichment’ gene sets. Those genes belonged to the leading-edge subsets and contributed the most to the enrichment scores, and are the most differentially expressed among the UPR genes. To visualize these results, we generated Venn diagrams using jvenn43, to display the common genes across alternative comparisons.\n\nIn order to verify the presence of unfolded protein response element (UPRE) and ER stress response elements (ERSE I and II)44 in the upstream regions (-1000bp to +500bp) of UPR genes upregulated in HD, we downloaded all the human promoter regions, (n=23322) available in the eukaryotic promoter database (EDP; http://epd.vital-it.ch)45 (RRID:nif-0000-02806). Next, we used Regulatory Sequence Analysis Tools (RSAT)46 to map these elements in the promoters and computed the enrichment of these stress elements in promoters of upregulated UPR genes compared to all the human promoters using hypergeometric test (equivalent to Fisher’s exact test).\n\nTo identify enriched biological processes in our gene sets we used BiNGO47 (RRID:nlx_149196) for Cytoscape48 (http://apps.cytoscape.org/apps/bingo) (RRID:nif-0000-30404); and GSEA42 (RRID:nif-0000-30629) to evaluate if genes from curated Reactome pathways (obtained from the Molecular Signature Database (MsigDB)49) were statistically over-represented. The significance of each identified biological process or pathway was calculated using the hypergeometric test, adjusted for multiple testing and converted to fdrs using the Benjamini and Hochberg method50 implemented in BiNGO (RRID:nlx_149196) or in GSEA (RRID:nif-0000-30629), respectively. We considered only those processes and pathways with an fdr of ≤ 0.05 to be significantly enriched.\n\n\nResults and discussion\n\nTo determine possible implications of the UPR in HD, we sought to assess its activation using a computational approach and the evaluation of existing data. First, we catalogued genes that are involved in UPR from several different sources and divided them into three different categories: UPR-KEGG-GO, UPR-interactions and UPR-literature as described in Material and Methods section and detailed in Table 1 and Supplementary Figure 1.\n\nSince changes in gene transcription are main effects of UPR activation and published microarray data are available for HD in humans as well as for HD models, we collected 12 different gene expression datasets generated for the study of HD: three datasets included expression from human blood and brain samples as well as human induced pluripotent stem cells (iPSCs); seven datasets were derived from HD mouse models and cell cultures; one from rat cells and one from yeast cells. If the expression dataset constituted time-series, we split the dataset according to the time points to maintain the temporal aspect of the expression changes.\n\nWe reasoned that UPR activity should be reflected in the regulation of UPR genes. By applying GSEA we tested whether UPR genes tend to be differentially expressed in HD samples or models compared to the corresponding controls. GSEA was employed, since it is able to detect modest but consistent tendencies in expression change within a pre-defined set of genes. This can be seen as a crucial advantage, as only small changes in gene expression are frequently observed in the study of neurodegenerative disease due heterogeneity of tissue samples and biological variability of the underlying processes.\n\nRemarkably, we found both indications for significant upregulation as well as repression of UPR genes in the different comparisons. Notably, significant differential expression was generally consistent across the three alternative UPR gene sets (with the exception of R6/1 mice at 27 weeks, where UPR-Literature genes tended to be downregulated while UPR-GO-KEGG genes displayed upregulation) (Figure 2). This observation implies that the obtained findings tend to be independent of the particular definition of UPR genes chosen in this study.\n\nFor each of the compiled UPR genes sets, the normalized enrichment scores (NES) produced by GSEA are shown for different comparisons of HD-associated expression with controls. Positive scores indicate a tendency towards upregulation; negatives scores indicate a tendency towards downregulation of genes in the UPR sets. Comparisons that showed significant upregulation of UPR gene sets (fdr ≤ 0.05 and NES ≥ +1.4) are highlighted by red background, while significant downregulation (fdr ≤ 0.05 and NES ≤ +1.4) by green background.\n\nFor most murine in vivo HD models, a significant upregulation was detected. Interestingly, the activation pattern was dependent on the time point of expression measurement for the two mouse models (R6/1 and YAC128), for which time series data were available. In the case of R6/1 mice, expressing exon 1 of the human HTT gene with a 115 CAG repeat, the most significant activation of UPR genes occurs after 18 and 22 weeks, while genes included in UPR-Literature and UPR-interactions sets tend to be downregulated after 27 weeks. In contrast, upregulation of UPR genes is only observed at the later time point (24 months), whereas downregulation dominates after 12 months for YAC128 mice containing the full length human HTT gene with 128 CAG repeats. This divergence may be explained by the rapid development of an aggressive disease phenotype in R6/1 mice compared to YAC128 mice, which show a milder phenotype with slower progression (Figure 2).\n\nInspecting the three human expression datasets, only the iPSC HD model showed a highly significant activation for all three UPR gene sets, whereas no consistent differential expression of the three UPR gene sets could be detected in blood and brain samples of HD patients. For whole blood samples, this observation might not be surprising, since erythrocytes - constituting the main component of blood - lack of an ER. The absence of a clear pattern in HD brain expression might be due to the fact that expression data were obtained from the post-mortem samples and thus represent typically only the very late stage of the disease. Finally, no significant alterations of expression was found for the rat in vitro and the yeast HD model (Figure 2).\n\nNext, we sought to identify UPR genes which showed conserved differential regulation in distinct HD models. For this purpose, we collected genes that were assigned to the enrichment core by GSEA in the comparisons that showed significant upregulation of UPR genes. In total we collected the UPR genes contained in the enrichment cores from five mouse HD models and the human HD iPSC model. Intersection of these sets led to the identification of 132 genes that were commonly upregulated, when UPR activation was indicated (Figure 3, Supplementary data file 1). We refer to this set of genes as UPR-HDup. As UPR has been also associated with the suppression or degradation of a substantial number of genes51,52, we carried out the equivalent procedure to identify UPR genes whose downregulation is conserved in different HD models. Here, we intersected the enrichments core from comparisons displaying suppression of UPR genes (Supplementary Figure 2, Supplementary data file 2). This resulted in 81 commonly downregulated genes. We refer to the combined set of UPR genes (consisting of 132 upregulated and 81 downregulated genes) that were commonly detected as differentially regulated in HD gene expression data as UPR-HDdiff.\n\nGenes included in the core enrichment set for comparisons that indicated UPR activation (highlighted in red in Figure 2) were compared. Common upregulated UPR genes (n=169) in five HD mouse models (left side) were intersected with upregulated UPR genes in human HD iPSCs (right side) resulting in a set of 132 UPR genes, whose activation was conserved across the different HD models. The bar plots (bottom) display the number of UPR genes that were assigned to the core enrichment sets for comparisons that indicated upregulation.\n\nTo assess whether the observed upregulation indeed reflects the activation of UPR or if it might be a consequence of other unrelated processes, we carried out an analysis of the promoter regions of genes included in UPR-HDup. We searched for the presence of sequence elements that indicate binding of transcription factors associated with the UPR. In particular, we searched for unfolded protein response elements (UPRE; TGACGTG (G/A)) and the alternative ER stress response elements I (ERSE I; CCAAT(N9)CCACG) and II (ERSE II; ATTGG-N-CCACG) in promoter regions (-1000 bp to +500 bp) regions of UPR-HDup genes. These characteristic sequence elements are targeted by the bZIP transcription factors ATF6 and XBP1, which are main mediators of the transcriptional adaptation evoked by UPR53.\n\nStrikingly, we found that the vast majority of the UPR-HDup has such a characteristic binding sequence in their promoter regions (Supplementary Figure 3). Compared to number of sequence elements that we would expect by chance, a highly significant overrepresentation was detected for the UPR-HDup genes. More specifically, we found the occurrence of UPRE in 104 genes (p=3.0∙10-9), ERSE-I in 93 genes (p=0.0019), and ERSE-II in 8 (p=0.052). Notably, a large number of UPR-HDup genes had alternative binding motifs included in the promoter region: 70 genes had both ERSE-I and UPRE, 2 genes had both ERSE-I and ERSE-II and six genes had all three elements (for list of genes see Supplementary data file 3) which might suggest that these genes are under particularly tight control of UPR-associated transcription factors ATF6 and XBP1. Altogether, the results of the promoter analysis support the conclusion that the upregulation of UPR genes in HD models faithfully reflects an activated UPR.\n\nSince the UPR comprises a complex series of diverse molecular mechanisms, we examined the functional composition of UPR-HDdiff genes. For this purpose, we performed functional analysis using BiNGO to identify enriched biological processes (as defined in GO) that are overrepresented among UPR-HDdiff genes. All the biological processes that are significantly enriched in our analysis are listed in Supplementary data file 4. Expectedly, we detected that stress-related functional categories such as ‘response to stress’ (GO ID:6950; n=44; fdr=2.08E-03) and ‘response to unfolded protein’ (GO ID:6986; n=13; fdr=4.55E-09) were enriched (Figure 4a). A second group of significantly overrepresented GO categories were related to ‘protein transport’ (GO ID:15031; n=34; fdr=1.76E-07) and ‘protein localization’ (GO ID:8104; n=36; fdr=1.19E-06) including ‘vesicle-mediated transport’ (GO ID:16192; n=24; fdr=9.03E-05) and ‘ER to Golgi vesicle-mediated transport’ (GO ID:6888; n=4; fdr=4.00E-02) (Figure 4b). Additionally, we also found ‘ER-nucleus signalling pathway’ (GO ID:6984; n=9; adjp-value=1.81E-07) to be highly enriched. It has been previously reported that ER-nucleus signalling pathway functions via activation of NF-ĸB due to ER-overload triggered by protein congestion54.\n\nGO hierarchies for biological processes overrepresented in the set of differentially regulated UPR are shown. Nodes indicate specific GO terms and their size represents the number of included UPR genes. The significance of overrepresentation (enrichment) is visualized by colour-coding from yellow to orange with the latter representing higher significance. No colour indicates that the process is not significantly enriched (fdr ≥ 0.05). The overrepresented biological processes can be split into four major themes: (a) response to stress, (b) protein transport, (c) protein catabolic process and (d) apoptosis.\n\nFurthermore, UPR-HDdiff genes tended to be associated with protein catabolism and in particular protein degradation (Figure 4c). Significant processes here were e.g. ‘protein catabolic process’ (GO ID:30163; n=18; fdr=6.40E-05), ‘proteasomal ubiquitin-dependent protein catabolic process’ (GO ID:43161; n=13; fdr=6.69E-06) and ‘protein ubiquitination’ (GO ID:31396; n=7; fdr=3.12E-02). These results coincide well with previous studies establishing the connection of UPR and ERAD and showing, for instance, that the extent of activation of the UPR is concurrent with the severity of ERAD defect55.\n\nFinally, genes linked to apoptosis could be found among the UPR-HDdiff genes (Figure 4d). Of particular interest for potential intervention could be genes associated with ‘regulation of apoptosis’ (GO ID:42981; n=24; fdr=1.65E-02), as their manipulation may prevent the execution of the apoptotic programme under persistent ER stress.\n\nIn summary, UPR genes detected as commonly differentially regulated in HD expression data were not restricted to a particular functional category, but can be associated with many processes linked to the UPR.\n\nComplementary to the functional composition, we evaluated whether specific pathways might be activated based on the observed commonly upregulated UPR genes (UPR-HDup). Therefore, we carried out pathway enrichment analysis using a set of pathways curated in the Reactome database. As expected, ‘unfolded protein response’ (n=6; fdr=2.04E-05), ‘activation of genes by ATF4’ (n=3; fdr=0.00291) and ‘PERK regulated gene expression’ (n=3; fdr=0.0033) were detected as significantly enriched among UPR-HDup genes (Figure 5). More interestingly, we also found an overrepresentation of components of the ‘immune system’ (n=14; fdr=6.74E-05), ‘adaptive immune system’ (n=6; fdr=1.96E-03), ‘NGF signalling’ (n=9; fdr=2.42E-03), and ‘Diabetes pathways’ (n=7; fdr=2.04E-05). Complete results from the analysis are included in Supplementary data file 5.\n\nThe network of pathways with overrepresentation of upregulated UPR genes are shown. Node size represents number of genes in each pathway and connecting line size represents number of genes shared between two pathways. Colour coding from white to red indicates increasing statistical significance. Pathways were derived from the Reactome database.\n\nRemarkably, recent studies have also suggested that ER stress and activated UPR are interconnected with inflammatory processes56. Inflammation is an immunological process usually carried out by the vascular system to counteract disease, and to fight foreign antigens against invasion. Within the brain, microglia and astrocytes play important immunological functions. Until very recently, little was known about inflammatory molecules in HD. Recent studies, however revealed a distinct profile of inflammatory mediators from post-mortem human HD tissue57,58. Inflammatory mediators such as IL-1β and TNF-α were increased only in the striatum, whereas IL-6, IL-8 and MMP-9 were also upregulated in cortex and in the cerebellum58. This supports the conjecture that secreted inflammatory cytokines and activated microglia cells could lead to axonal damage and extensive neuronal cell death in HD pathology57–60. In general, activated microglia exert their diverse effects on neurons and macroglia (astrocytes and oligodendrocytes). Inflammation occurs through the release of cytoprotective agents such as growth factors, plasminogen, and neuroprotective cytokine as well as cytotoxic substances such as oxygen radicals, nitricoxide, glutamate, proteases, and neurotoxic cytokines. One of the earliest reports describing microglial abnormalities in HD was provided by Singhrao et al.60. Microglial cell counts were considerably increased in the caudate putamen of HD and these microglial cells expressed increased amounts of complement factors. A more detailed investigation of microglial morphological changes associated with HD was performed by Sapp et al.61. The authors localized morphologically activated microglial cells in the neostriatum, cortex and globus pallidus as well as in adjoining white matter of HD brains. Additionally, positron emission tomography (PET) studies using the ligand for benzodiazepine receptor (PK-11195), which labels activated microglia have been employed to study of neuroinflammation. Using this technique, Tai et al.62,63 demonstrated that microglial activation in HD patients correlates with disease progression as assessed by loss of dopamine D2 receptor binding sites. Interestingly, Tai et al. could also demonstrate that microglial activation and release of cytokine IL-6 is observed in presymptomatic HD gene carriers and can be detected up to 15 years before predicted age of onset. These findings indicate the microglial inflammatory activation is an early event associated with subclinical progression of HD and may constitute a target for early therapeutic intervention.\n\nBesides the indication of processes related to the immune response, results of the pathway enrichment analysis also pointed to diabetes. It has been shown that diabetes in Wolcott-Rallison syndrome (a rare autosomal recessive form of juvenile diabetes) is a result of high levels of ER stress caused by mutations in the PERK gene in pancreatic β-cells. In addition, studies have shown that HD patients show increased incidence of diabetes64,65 and HD transgenic mice develop hyperglycemia66,67. More recently it has been experimentally validated that HD transgenic mice develop intranuclear inclusions in the pancreatic β-cells, causing an intrinsic defect in insulin production68.\n\nTo narrow down the list of UPR-HDdiff genes for further inspection, we utilized additional information, including a reference set of potential molecular targets for HD therapy that were made available through the HD Research Crossroads database initiated by the CHDI Foundation (see Kalathur et al. 2012)37. Genes were included by experts in the field based on the evaluation of published literature and in-house screens using a set of defined criteria (see Kalathur et al. 2012)37. For instance, a gene was considered as a potential HDTT if genetic or pharmacologic modification of its activity led to a change of a HD-related phenotype in a validated cell culture or organism model of HD. At present, this curated reference set constitutes the most comprehensive collection of HDTTs. In addition, we extracted genes, whose corresponding proteins were reported to be physically associated with HTT, from the HDNetDB database. We recently demonstrated that HTT interactors tend to be enriched in proteins that influence the toxicity of mHTT, and provide favourable candidates for the identification of molecular modifiers of HD69.\n\nWe reasoned that differentially regulated UPR genes, which have been shown to influence HD-related phenotypes and to be physically associated with HTT, could constitute molecular links between UPR and HD. Therefore, we integrated the three gene lists and identified 13 genes that were common to all three: RAB5A, HMGB1, CTNNB1, DNM1, TCP1, TUBB, TSG101, DNAJB1, CCT2, EEF2, DYNC1H1, HSPA5 and SLC12A5 (Supplementary Figure 4). Notably, the search for stress response elements in the upstream regions of these 13 genes revealed that eight genes (RAB5A, HMGB1, CTNNB1, DNM1, TCP1, TUBB, TSG101 and DNAJB1) possess either UPRE or ERSE or both elements in their promoters, suggesting that these genes are under direct control of UPR-associated transcription factors (Table 3).\n\n+ indicates the presence of particular stress response element in the promoter regions (+1000 to -500 bp).\n\nInspection of the genes possessing UPRE or ERSE elements in their promoter regions revealed that four of them (TCP1, CCT2, DNAJB1 and HMGB1) have been reported to act as chaperones. Besides being essential components of the UPR, molecular chaperones can modulate the aggregation and toxicity of proteins, including mHTT. TCP1 (CCT1) and CCT2 are components of the TCP1 ring complex (TRiC) that uses cycles of ATP-binding and hydrolysis to bind unfolded polypeptides and facilitate their folding. Notably, TRiC has been identified as a potent suppressor of mHTT mediated toxicity and inhibitor of the mHTT protein aggregation in vitro and in vivo70. DNAJB1 belongs to the group of DnaJ/Hsp40 (Heat shock protein 40) proteins that are involved in protein translation, folding and translocation through regulating ATPase activity of the Hsp70s chaperones. In a PC12 cell model, experiments indicated that DNAJB1 attaches to soluble mHTT oligomers and recruits Hsp70 suppressing mHTT mediated toxicity71. Finally, HMGB1 encodes for the High-mobility group box 1 protein (HMGB1), which has recently been demonstrated to have chaperone-like activity, inhibiting aggregation of various proteins. Overexpression of HMGB1 can also decrease the aggregation induced by extended polyQ stretches72.\n\nA crucial aspect of the UPR in the context of HD is the possibility that it can trigger apoptosis upon persistent ER stress. To obtain a comprehensive view of the connections between UPR and apoptosis, we applied a network approach. First, we generated the UPR interactome from known protein interactions of UPR core components, which we extracted from the UniHI and HDNetDB databases (Supplementary data file 6). Second, we compiled a list of genes (n=594) associated with apoptosis from several different sources (as described in the materials and methods). We then used this list to identify 40 proteins associated with apoptosis within the UPR interactome (Supplementary data file 7). These genes included, among others, Apoptosis Signal Regulating Kinase 1 (ASK1, also known as MAP3K5), whose knock-out in primary neuron provided protection from ER stress-induced JNK activation and cell death triggered by polyQ fragments73.\n\nAs the mutation in HTT can perturb the function of interacting proteins by aberrant binding, we checked for each of the 40 proteins whether they have been reported to physically associate with HTT. Using molecular interaction data collected in HDNetDB, we detected that six of the 40 proteins interact with HTT i.e. ADD1, HSP90B1, IKBKB, RPS3A, IKBKG and LMNB1 (Supplementary data file 7). A visualisation of the UPR interactome with apoptosis-related proteins and HTT interactors highlighted can be found in Figure 6.\n\nThe network displays UPR proteins and their interactions. Nodes indicate proteins and lines represent molecular interactions between them as derived from UniHI and HDNetDB. UPR proteins which are also associated with apoptosis are highlighted in red, while UPR proteins that are both associated with apoptosis and interactors are in green.\n\nLiterature review showed that the two proteins kinases IKBKB and IKBKG, the laminin LMNB1 and the ribosomal protein RPS3A have been previously linked to neurodegenerative diseases. IKBKB and IKBKG are subunits of IkB kinase (IKK). They activate members of the NF-ĸB transcription factor family by phosphorylation of their inhibitor (IkB)74 leading to ubiquitination and destruction of IkB, thereby allowing activation of the NF-ĸB complex. NF-ĸB maintains the balance between cell survival and apoptosis75. Although unrelated to ER stress, it has been shown that inhibition of IKBKB decreases HTT proteolysis in a cell model, and thus might lower the load of toxic HTT fragments in HD76. Recently, it has been reported that ubiquitination of IKBKG by Parkin, an ubiquitin ligase associated with Parkinson’s disease regulates the anti-apoptotic pathway that is key to maintaining mitochondrial integrity77.\n\nLamin B1 protein, LMNB1 is thought to be involved in nuclear stability and chromatin structure. Experiments in Caenorhabditis elegans overexpressing aggregation-prone peptides identified laminins as modulators of protein toxicity at neuromuscular junctions78. Further, in leukodystrophy mouse models, lamin B1 acts as an important regulator of myelin formation and maintenance79, in humans lamin B1 gene duplications80 and large deletions upstream of promoter regions can cause autosomal-dominant leukodystrophy81. More importantly, a recent study reports increased levels of lamin B1 in both human HD patients and the R6/1 mouse model of HD82. Due to the involvement of lamin B1 in several cellular alterations such as chromatin organisation, gene transcription and proteotoxicity, alterations in lamin B1 expression might have important implications in HD pathophysiology.\n\nFinally, it has been demonstrated that apoptosis is induced by inhibiting the expression of ribosomal protein S3A (RPS3A)83. It also has been observed that SNP variants in RPS3A homologues are associated with pathogenesis of Alzheimer’s disease84. Apart from its function as a ribosomal protein, RPS3A might also act as a chaperone. Co-expression of mouse RPS3A suppressed the toxicity induced by α-synuclein (which is a major components of Lewy bodies observed in Parkinson’s disease) in a yeast model system85.\n\nAs the literature review indicated, the intersection of the UPR interactome with apoptosis-related genes and HTT interactors can point out proteins with potential relevance for neurodegeneration. Thus, the generated gene lists provided in the Supplementary data file 6 and Supplementary data file 7 might give interested researchers a valuable basis for more detailed inspections.\n\n\n\n\nConclusions\n\nVarious studies have indicated a role of the UPR in HD. However, its relevance for therapeutic interventions remains to be established. With the presented work, we aimed to delineate the connection between UPR and HD by examining available HD-relevant gene expression and molecular interaction data. We found indications for differential regulation of UPR genes in a number of expression studies. Notably, the observed differential regulation is not conserved across all evaluated studies reflecting the well-known heterogeneity of current HD models. This needs to be taken into account for future studies of the UPR in the context of HD. The results of our analysis (displayed in Figure 2) may therefore serve as guidance for the choice of model systems. Despite the observed heterogeneity, the comparison nevertheless indicated a number of genes that tend to be commonly regulated in different expression studies. This finding enabled us to define core sets of UPR genes that were commonly up- or downregulated in different studies. This derivation was supported by the detection of a significant overrepresentation of UPR-associated stress response elements (UPRE and ERSE) in the promoter regions of the upregulated genes.\n\nFunctional enrichment analysis on differentially expressed UPR genes pointed to a broad range of mechanisms involved. Additional pathway analyses indicated the activation of inflammatory processes and a potential connection to diabetes. Including complementary data sets, we identified UPR genes that have been indicated to influence HD pathogenesis. Finally, we derived sets of genes that connect UPR with apoptosis and might be directly influenced by mHTT.\n\nIn summary, through our work we present the first comprehensive analysis of UPR activation in HD and elucidate potential links to pathogenetic mechanisms within a systems biology framework. While our work cannot provide definite proofs for the identified relations due to its purely computational nature, it can nevertheless constitute a broad basis for experimental follow-up investigations. To assist such endeavours, extensive supplementary material has been provided together with this article with the aim of helping independent researchers to select genes of interest. We are also currently developing a publicly accessible web-portal for the retrieval and visualisation of changes in UPR-associated gene expression across the evaluated transcriptomics studies. In conclusion, we hope that our work can contribute to a better understanding of the UPR in HD and eventually to the identification of novel therapeutic targets to cure HD.\n\n\nData availability\n\nFigshare: Raw data for Kalathur et al., 2015 ‘The unfolded protein response and its potential role in Huntington’s disease elucidated by a systems biology approach’ doi: 10.6084/m9.figshare.138308087", "appendix": "Author contributions\n\n\n\nRK collected the data, performed the analysis and prepared manuscript. JGL, SM and KA collected data and wrote parts of manuscript. MF conceived the study, contributed to interpretation of the results and wrote the final version of the manuscript. All authors agreed to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe work presented was supported by CHDI Foundation (A-2666) and by the Portuguese Fundação para a Ciência e a Tecnologia (SFRH/BPD/70718/2010 to RKK and IF/00881/2013 to MEF).\n\n\nAcknowledgements\n\nWe would like to thank Isabel Duarte for critical reading of the manuscript.\n\n\nSupplementary information\n\nVenn diagram comparing the three different UPR genes lists generated.\n\nComparison of several HD expression data sets to identify UPR genes that are downregulated in different HD models.\n\nVenn diagram showing common genes between 3 data sets, UPR-HDdiff: UPR genes that are differentially regulated in HD; HDTT: HD therapeutic targets as described by Kalathur RK et al.37 and originated by the HDCrossRoads database; and HTT-int: HTT interactors derived from the HDNetDB.\n\n\nReferences\n\nA novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington’s disease chromosomes. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPadiath QS, Saigoh K, Schiffmann R, et al.: Lamin B1 duplications cause autosomal dominant leukodystrophy. Nat Genet. 2006; 38(10): 1114–23. PubMed Abstract \| Publisher Full Text\n\nGiorgio E, Robyr D, Spielmann M, et al.: A large genomic deletion leads to enhancer adoption by the lamin B1 gene: a second path to autosomal dominant adult-onset demyelinating leukodystrophy (ADLD). Hum Mol Genet. 2015; pii: ddv065. PubMed Abstract \| Publisher Full Text\n\nRue L, Alcala-Vida R, Lopez-Soop G, et al.: Early down-regulation of PKCδ as a pro-survival mechanism in Huntington’s disease. Neuromolecular Med. 2014; 16(1): 25–37. PubMed Abstract \| Publisher Full Text\n\nNaora H, Takai I, Adachi M: Altered cellular responses by varying expression of a ribosomal protein gene: sequential coordination of enhancement and suppression of ribosomal protein S3a gene expression induces apoptosis. J Cell Biol. 1998; 141(3): 741–53. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGrupe A, Li Y, Rowland C, et al.: A scan of chromosome 10 identifies a novel locus showing strong association with late-onset Alzheimer disease. Am J Hum Genet. 2006; 78(1): 78–88. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDe Graeve S, Marinelli S, Stolz F, et al.: Mammalian ribosomal and chaperone protein RPS3A counteracts α-synuclein aggregation and toxicity in a yeast model system. Biochem J. 2013; 455(3): 295–306. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim I, Xu W, Reed JC: Cell death and endoplasmic reticulum stress: disease relevance and therapeutic opportunities. Nat Rev Drug Discov. 2008; 7(12): 1013–30. PubMed Abstract \| Publisher Full Text\n\nKalathur R, Giner-Lamia J, Machado S, et al.: Raw data for Kalathur et al., 2015 ‘The unfolded protein response and its potential role in Huntington ́s disease elucidated by a systems biology approach’ Figshare. 2015. Data Source" }	[ { "id": "9713", "date": "29 Jul 2015", "name": "Scott Zeitlin", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 HD and other neurodegenerative disorders involving the accumulation of misfolded protein, ER stress and the activation of UPR have been implicated in pathogenesis, although the extent to which it contributes to neuronal cell death is still unclear. In this paper, the authors employ a bioinformatic approach to show that the unfolded protein response (UPR) is activated in a variety of human and animal Huntington’s disease (HD) models. It is noteworthy that the authors also demonstrate that the upregulation of the UPR genes is most likely a direct response to UPR activation by analyzing the promoter regions of these genes for the binding sites of the UPR transcriptional activators ATF6 and XBP1. Moreover, the authors also identified sets of genes that provide a potential link between both UPR and HD and between UPR and apoptosis. Pathway enrichment analysis was also used to identify functional pathways activated by the common set of upregulated UPR genes. In addition to the expected pathways associated with UPR (e.g. PERK-regulated gene expression and ATF-activated genes), the authors also identified components of the immune system, neurotrophin signaling, and diabetes. Interestingly, there is evidence in the HD literature that all three of these latter pathways are affected in HD.This work is a good example of the power of using a systems biology approach to provide a fairly comprehensive analysis of UPR activation in HD. The authors are appropriately cautious in emphasizing that their results do not prove that the pathways and relationships between pathways that they have identified all contribute to HD pathogenesis. However, their results provide an excellent guide for further experimental studies, and the authors’ development of a publicly accessible web site for the retrieval and visualization of their UPR-associated gene expression data in HD will be an important tool for the field that will facilitate these future studies. Minor comments:In the introduction, the authors mention that although HTT is not located in the ER, there are there are several potential mechanisms by which mutant HTT could induce ER stress (impairment of ERAD, dysfunctional vesicular trafficking, and altered ER calcium homeostasis). Atwal et al., (2007) and Atwal and Truant (2008) have shown that there is a more direct link between HTT and ER stress. In their work, they show that the N17 domain of HTT is a stress-sensitive ER association domain, and that the expanded polyQ stretch in mutant HTT perturbs the release of mutant HTT from ER and its translocation of HTT into and out of the nucleus in response to cell stress events. p.7, 1st paragraph: “…UPR has been also associated with the suppression or degradation of a substantial number of genes…. Do the authors mean: …UPR has been also associated with the suppression or degradation of a substantial number of genes or gene products? To account for the differences in differential UPR gene expression changes that were observed among the different HD mouse models, the authors suggest that differences in the HTT transgenes and their expression levels among the models could be responsible. In addition, the different genetic backgrounds of the models can contribute to the variability. In future studies aimed at studying the role of the length of mutant HTT’s expanded polyQ stretch in UPR activation, the authors may also want to consider evaluating CHDI’s publicly available transcriptome data obtained from the cortex and striatum of 6-month old knock-in HD mouse models expressing wild-type or mutant Htt alleles with different CAG repeat lengths.", "responses": [ { "c_id": "1818", "date": "02 Mar 2016", "name": "Ravi Kiran Reddy Kalathur", "role": "Author Response", "response": "We would like to thank the reviewer for the encouraging evaluating of our systems biology approach and his suggestions for further improvement of the study and its presentation. In response to the comments, following changes to the manuscript were made:In the Introduction, we pointed out the finding of a stress-sensitive association of HTT with the ER membrane, which suggests a potential direct role of HTT in ER stress. The relevant references are now included. We clarified the meaning of the sentence. It states now: “As the UPR has also been associated with suppression of gene transcription and the enhanced degradation of numerous transcripts, ...” We would like to thank reviewer for pointing out this data set. We analysed the correlation of expression changes with the length of polyglutamine tract. Notably, UPR genes were found to be strongly overrepresented among genes whose expression in the striatum significantly correlate with the length of polyglutamine tract. The correlation and the corresponding significance are also shown for the potential UPR-HD connectors in table 3 and Supplementary dataset 9. The results suggest length of the polyglutamine tract (which is reversely correlated with age of disease onset in human patients) plays a critical role in the activation of UPR. We aim to include these expression changes also in our newly developed web-portal UPRHD at http://uprhd.sysbiolab.eu." } ] }, { "id": "9517", "date": "05 Aug 2015", "name": "Stefan Taubert", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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, Kalathur et al. investigated the role of the ER's unfolded protein response (UPR) in the disease pathogenesis of Huntington's Disease (HD). Although HTT, the protein mutated in HD, does not localize to the ER, mutated HTT intereferes with downstream UPR processes, which may prevent the UPR from functioning normally and thus result in the induction of apoptosis by the UPR, which in turn could underlie the dramatic neuronal loss observed in HD. To broadly evaluate the role of UPR activation in the many experimental HD models and contexts, the authors used a bioinformatics approach to query for the role of the UPR in HD pathology, assessing transcriptomes, regulatory DNA elements, and UPR interactomes. The resulting data point to a core set of UPR genes that were commonly (although, as nicely pointed out, not universally) up- or downregulated in distinct experimental HD models, pro-inflammatory events involving microglia, a putative link between HD and diabetes/hyperglycemia, and the identification of a core set of genes that link UPR to apoptotic signaling and that mHTT may impinge upon.Overall, this is a nice body of work. This computational analysis is comprehensive and multifaceted, the data interpretation measured and well-qualified, the paper well written, and the data are very accessible. One of the key strengths is that the authors aimed to integrate data obtained in many diferent experimental HD systems - various mouse models, human data, and even yeast and rat models. This comprehensive approach allows them to point to evolutionarily conserved genes and processes as suitable candidates for future investigation.Specific criticism:To derive their set of HTT interacting proteins, the authors use a dataset downloaded from HDNetDB database, which yields a large set of HTT interactors (HTT-int) including 1015 genes, including, as acknowledged, presumably many indirect interactors. Perhaps a deeper up-front curation for putative direct interactors would have been useful. In Figure 2, the authors assess the overlap between genes regulated in 6 mouse HD datasets and one human HD. To ensure best stringency, the first limit the mouse dataset to those genes that are regulated in all 6 individual datasets. While this high stringency is laudable, one could have also lowered the stringency a bit in regards to inclusion in the core mouse IPR set, i.e. inclusion in 6/6 sets was deemed required, but expanding the set to 5/6 or even 4/6, resulting in 420 and 902 additional genes, respectively, would have only marginally lowered the stringency while providing a larger set for the determination of evolutionary conservation.", "responses": [ { "c_id": "1817", "date": "02 Mar 2016", "name": "Ravi Kiran Reddy Kalathur", "role": "Author Response", "response": "We thank the reviewer for the positive comments and specific suggestions that helped us to further improve our manuscript. In the revised version, we have included the additional analysis suggest by the reviewer.We distinguish now between direct and indirect interactions of HTT based on their annotation in HDNetDB. An additional supplementary table (Supplementary dataset 8) presents now the classification of HTT interactions. This information was also used for the interpretation of our results. For instance, 2 of the 13 proteins constituting a putative link between HD and UPR, were direct interactors. We did not want to exclude a priori all indirect HTT interactors, since those might be part of larger protein complexes with HTT and thus potentially important for the HTT’s molecular functions. Additional lists of genes with evolutionary conserved expression were generated for less stringent filters (i.e. requiring the inclusion in the enrichment cores of at least 5 or 4 comparisons for up- or downregulated genes, respectively). In general, promotor and enrichment analyses of the extended gene lists agree well with those for the more stringent genes lists. The results are presented in supplementary tables and figures, and briefly discussed in the main manuscript. For the connection to HD, lowering the requirement led to detection of 53 candidates, of which 15 were direct HTT interactors (Supplementary figure 5). This enlarged set is presented in Supplementary dataset 9 for the interested reader." } ] } ]	1	https://f1000research.com/articles/4-103
https://f1000research.com/articles/5-250/v1	01 Mar 16	{ "type": "Antibody Validation Article", "title": "Validation of a commercially available anti-REDD1 antibody using RNA interference and REDD1-/- mouse embryonic fibroblasts", "authors": [ "Deborah L. Grainger", "Lydia Kutzler", "Sharon L. Rannels", "Scot R. Kimball", "Lydia Kutzler", "Sharon L. Rannels" ], "abstract": "REDD1 is a transcriptional target gene of p53 and HIF-1, and an inhibitor of mTOR (mechanistic target of rapamycin) complex 1 (mTORC1)-signaling through PP2A-dependent interaction, making it an important convergence point of both tumor suppression and cell growth pathways. In accordance with this positioning, REDD1 levels are transcriptionally upregulated in response to a variety of cellular stress factors such as nutrient deprivation, hypoxia and DNA damage. In the absence of such conditions, and in particular where growth factor signaling is activated, REDD1 expression is typically negligible; therefore, it is necessary to induce REDD1 prior to experimentation or detection in model systems. Here, we evaluated the performance of a commercially available polyclonal antibody recognizing REDD1 by Western blotting in the presence of thapsigargin, a pharmacological inducer of ER stress well known to upregulate REDD1 protein expression. Further, REDD1 antibody specificity was challenged in HEK-293 cells in the presence of RNA interference and with a REDD1-/- mouse embryonic fibroblast knockout cell line. Results showed reproducibility and specificity of the antibody, which was upheld in the presence of thapsigargin treatment. We conclude that this antibody can be used to reliably detect REDD1 endogenous expression in samples of both human and mouse origin.", "keywords": [ "DDIT4", "mTOR", "REDD1", "thapsigargin" ], "content": "Introduction\n\nMechanistic target of rapamycin complex 1 (mTORC1) is a central signaling node in the cellular response to nutrient availability and growth factor signaling, initiating and regulating processes such as protein synthesis, ribosome biogenesis and de novo lipogenesis when active (reviewed by Laplante and Sabatini1). However, in times of amino acid deprivation or energy deficit, the cell must switch from these anabolic processes to maintain energy homeostasis. The switch is controlled by changes in mTORC1 activity that occur in response to variations in the availability of nutrients and energy requirements of the cell (see recent review by Albert and Hall2).\n\nREDD1 (protein regulated in development and DNA damage response 1) also known as DDIT4 (DNA damage-inducible transcript 4 protein) or RTP801, is a 232 amino-acid upstream repressor of mTORC1 activity3–5 that is transcriptionally upregulated by growth factor signaling and in response to amino acid deprivation, among other stimuli. The mechanism by which REDD1 acts to repress mTORC1 signaling has been under investigation for almost a decade6. These studies are focused on REDD1’s suppression of mTORC1 via its stimulation of the tuberous sclerosis complex 2 (TSC2); however, the method by which REDD1 activates TSC2 remained elusive6.\n\nRecent data have revealed a model where REDD1 promotes the association of protein phosphatase 2A (PP2A) with serine/threonine-protein kinase Akt, leading to the dephosphorylation of the kinase on Thr3086. This dephosphorylation of Thr308 (but not the Ser473 residue) subsequently leads to a reduction in the Akt-mediated phosphorylation of TSC2, followed by TSC2-mediated stimulation of Rheb GTPase activity. This results in accumulation of Rheb in the GDP bound form, and thus the inhibition of mTORC1 activity.\n\nDespite its ubiquitous distribution, expression of REDD1 is typically negligible in developed tissues until cells encounter conditions of nutrient and energy deprivation7,8 or – particularly in the case of skeletal muscle tissue – during endurance exercise9 when energy is prioritized for movement. It is also transcriptionally activated by p5310 hypoxia inducible factor-1 (HIF-1)11 and ATF412 transcription factors, in accordance with initial findings that REDD1 levels increase in response to a variety of cellular stresses, including hypoxia3, ER stress12 and by agents that cause DNA damage such as UV radiation13. Several pharmacological agents can also be used to induce REDD1 expression in experimental models where REDD1 follows endogenous patterns of expression. Agents such as the glucocorticoid dexamethasone14 and the non-competitive inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pump thapsigargin12 have been shown to upregulate expression of REDD1.\n\nWe examined Proteintech’s anti-REDD1 antibody (catalog number 10638-1-AP) in scenarios where REDD1 levels were either reduced or absent. The antibody, which is raised against a whole-fusion protein immunogen consisting of amino acid residues 1-232, is cited in over 90% of publications utilizing REDD1 antibodies (CiteAb: http://www.citeab.com/search?q=REDD1), and a total of 110 publications (at the time of writing, http://www.ptglab.com/Products/REDD1-Antibody-10638-1-AP.htm?publication=1). REDD1 reduction was achieved using RNA interference (RNAi). Antibody specificity was further supported by Western blotting experiments performed using samples from a REDD1-/- knockout mouse embryonic fibroblast (MEF) cell line, and controlled in the presence of thapsigargin.\n\n\nMaterials and methods\n\nThe anti-REDD1 antibody used in this study (Cat#: 10638-1-AP, RRID: AB_2245711) is a rabbit polyclonal antibody made exclusively by Proteintech Group. It was raised against a fusion protein corresponding to the full-length human REDD1-protein sequence (amino acids 1-232). Full sequence: MPSLWDRFSSSSTSSSPSSLPRTPTPDRPPRSAWGSATREEGFDRSTSLESSDCESLDSSNSGFGPEEDTAYLDGVSLPDFELLSDPEDEHLCANLMQLLQESLAQARLGSRRPARLLMPSQLVSQVGKELLRLAYSEPCGLRGALLDVCVEQGKSCHSVGQLALDPSLVPTFQLTLVLRLDSRLWPKIQGLFSSANSPFLPGFSQSLTLSTGFRVIKKKLYSSEQLLIEEC.\n\nAll validations in this study were undertaken using lot# 00019207.\n\nA protein BLAST search demonstrates that the full-length sequence of human REDD1 shares over 90% homology with mouse Redd1.\n\nGoat anti-Rabbit polyclonal secondary antibody, conjugated with horseradish peroxidase (HRP) was obtained from Jackson ImmunoResearch (Cat# 111-035-003, RRID: AB_2313567). This antibody is specific for both the heavy and light chains of Rabbit IgG, and was affinity purified to reduce cross-reactivity to other species.\n\nThe anti-α-tubulin antibody used in this study (sc-32293, RRID: AB_628412) is a mouse monoclonal antibody purchased from Santa Cruz Biotechnology, Inc. The antibodies used in this study are summarised in Table 1.\n\nHEK-293 cells were purchased from the American Type Culture Collection (ATCC CRL-1573) and used in shRNA knock down validation studies of the 10638-1-AP anti-REDD1 antibody.\n\nThe REDD1-/- knockout MEF cell line and permission for its use was obtained from Dr. Leif Ellisen. Construction of the REDD1-/- allele and generation of this cell line was previously described by Sofer et al.1.\n\nDMEM high-glucose medium was purchased from Gibco/Life Technologies (11965-092) and Fetal Bovine Serum Premium Select was purchased from Atlanta Biologicals (51150).\n\nThe target site for REDD1 knock down was determined by available literature and online shRNA design tools (Broad Institute: http://www.broadinstitute.org/rnai/public/seq/search, siDirect version 2.0: http://sidirect2.rnai.jp/). We designed two single-stranded 21mer stem DNA oligonucleotides, encoding the target siRNA (sense strand), in addition to their single-stranded complementary strand counterparts (antisense strand). The two sequences (sense and antisense) were linked together by a short linker sequence TTCAAGACG that forms a hairpin loop structure upon sequence expression. At the end of the shRNA template we added a 6 nucleotide poly (T) tail, recognized as a termination signal. The 5’ ends of the two oligonucleotides are non-complementary and form the BamHI and HindIII restriction site overhangs which facilitated directional cloning into the pGenesil-1 vector. pGenesil-1 is a plasmid vector modified by addition of the hU6 promoter to the pEGFP-C1 plasmid. The sense strand sequences of both shRNA constructs designed for this study are shown in Table 2.\n\nHEK-293 cells (2×105) were seeded in fresh, full media in duplicate per experimental condition in 12-well CellBind plates (Corning). Cells were incubated for 24 hours at 37°C in a humidified incubator (5% CO2) prior to REDD1 knock down. Lipofectamine 2000 (Life Sciences, #11668027), a cationic lipid based transfection reagent, was mixed with pGenesil-1 vector containing REDD1 shRNA or control shRNA construct at a ratio of 1:1 in reduced serum medium and incubated for 15 minutes at 37°C to allow formation of lipid-DNA complexes. Cell culture media in 12-well plates were replaced with fresh pre-warmed media lacking serum before addition of lipid-DNA complex to HEK-293 cells. Cells were incubated with lipid-DNA complex for 48 hours at 37°C (5% CO2), before removal of media and washing in PBS. Cells were then harvested and lysed by application of 1X Laemmli sample buffer and scraping. REDD1 levels were detected by Western blot experiment.\n\nREDD1-/- and wild type cells were seeded at 2.5×105 in 12 well dishes in DMEM high glucose media containing 10% fetal bovine serum and 1% penicillin/streptomycin (Life Technologies #15070-063). Cells were incubated for at least 24 hours at 37°C in a humidified incubator with 5% CO2 prior to treatment. The medium was removed and replaced with medium containing either 100 nM thapsigargin (Sigma #T9033) or vehicle (ethanol), and the cells were returned to the incubator for 4 hours. The medium was then removed, cells were washed once with cold PBS, and harvesting and lysis by application of 1X Laemmli sample buffer and scraping.\n\nCell lysates were heated at 100°C for 5 minutes, before 40 µl lysate (per sample) was loaded onto a 4–20% Criterion SDS-polyacrylamide gel (BioRad). Proteins were separated by SDS-PAGE (200V for 1 hour). Separated proteins were transferred to PVDF membrane in a Criterion Blotter (BioRad) at 50V for 90 min. Membrane was blocked in 5% milk in TBS-Tween (TBS-T) for one hour before addition of primary antibody (1:800 dilution) in 1% milk TBS-T. Primary antibody incubation was carried out overnight before membrane washing in TBS-T and addition of secondary antibody (1:7,500 dilution) in 5% milk TBS-T for a further hour. Excess secondary antibody was removed by washing in TBS-T, before Protein bands were visualised by incubation with Clarity Western ECL Blotting Substrate (BioRad) and imaging on a FluorChem M imaging system (ProteinSimple). All steps were carried out at room temperature and are listed Table 3. Western blot signals were also further analysed by densitometry analysis using Alphaview (ProteinSimple).\n\n\nResults\n\nWe reduced endogenous levels of REDD1 using a homemade shRNA construct designed to target REDD1 mRNA (as detailed in the methods section). An initial RNAi experiment was carried out using 1.6 µg REDD1 shRNA plasmid or control shRNA plasmid to transfect HEK-293 cells. Cells were incubated with shRNA plasmid for 48 hours before cell lysis. REDD1 levels were then immediately visualized by Western blot experiment using the Proteintech anti-REDD1 antibody (10638-1-AP).\n\nWe found that REDD1 expression was reduced by up to 58% in cells incubated with REDD1-targeting shRNA (this percentage was obtained using data from REDD1-2 shRNA samples, n=2) compared to cells transfected with the control plasmid as shown in Figure 1a (assessed by densitometry measurements normalised to alpha-tubulin levels, as shown in Figure 1b). To further observe whether a higher percentage of reduction could be achieved by increasing the amount of shRNA, a second experiment using 3.2 µg REDD1 shRNA plasmid was performed. However, a further reduction of REDD1 levels was not observed (results not shown).\n\nREDD1 was detected using Proteintech’s 10638-1-AP anti-REDD1 antibody (A). Signal intensity was measured by densitometry analysis and blotted relative to α-tubulin control signal, n=2 (B).\n\nA second experiment was set up to further probe the specificity of Proteintech’s REDD1 antibody. REDD1-/- knockout and REDD1+/+ MEF cells were incubated with thapsigargin or carrier control before protein lysate preparation and subsequent Western blotting.\n\nIn accordance with Proteintech’s REDD1 antibody being highly specific for REDD1, the Western blot results show that REDD1 signal is present only in REDD1+/+ MEF cells and increase in response to thapsigargin. No such response was seen in the REDD1-/- knockout MEFs regardless of thapsigargin treatment. This observation was seen in three independent experiments (n=3); the data shown in Figure 2 are representative and were obtained following a 20 minute exposure of the Western blot membrane.\n\nREDD1 bands were indirectly detected using Proteintech’s 10638-1-AP anti-REDD1 antibody (n=3). Membrane exposure = 20 minutes. Numbers and block arrows indicate positions of 37 kDa and 25 kDa bands of the MW marker.\n\n\nConclusion\n\nThrough testing Proteintech’s anti-REDD1 antibody (10638-1-AP) in a REDD1-/- knockout (KO) MEF cell line, and to a certain extent, by using it in combination with REDD1 shRNA plasmids in HEK-293 cells, we have demonstrated a loss or reduction of REDD1 protein signal on Western blot membranes, respectively – indicative of antibody target specificity. Antibody signal also responds accordingly to the presence of thapsigargin-treated MEF cell lines. This paper validates the specificity of this commercially available antibody for the REDD1 protein in Western blots, supporting a significant prior body of work that has utilised this immunological reagent.\n\nThere are several observations we must address in this conclusion; the first being REDD1’s observed molecular weight (MW) on Western blot membranes. Despite having a predicted MW of 25 kDa, REDD1 often migrates around 35 kDa during SDS-PAGE due to the presence of multiple lysines in its amino acid sequence, as documented by other sources10,16,17. The MW seen for REDD1 in our investigations are in concordance with the reported MW shift. Second, the faint bands present in Figure 2 could perhaps warrant further investigation, but as the blot shown represents a prolonged membrane exposure (20 minutes), these artefacts are unlikely to cause misinterpretation of results in experiments, given the signal intensity in the stimulated wild type control (WT+).\n\nThe level of REDD1 knock down in the RNAi experiment was lower than desired (calculated to be around a 58% reduction of REDD1 levels). However, given the loss of REDD1 signal in the Western blots featuring REDD1-/- knockout (KO) MEF cells – a cell line well-characterised by previous studies6,15,18–20 – using the same antibody lot as in the knock down experiment, we feel there is enough evidence to confirm REDD1 specificity.\n\nAt the time of paper submission, the #00019207 lot was the only lot available from the vendor. Lot-to-lot testing is carried out by the vendor, but not yet in the context of REDD1 absence or knock down (though data of an initial RNAi experiment appear on the online datasheet, they represent the same lot #00019207); therefore it would be interesting to follow up this study in future by testing further lots of the 10638-1-AP REDD1 antibody in such settings where REDD1 levels are absent or compromised.\n\n\nData availability\n\nF1000Research: Dataset 1. Raw data for ‘Validation of a commercially available anti-REDD1 antibody using RNA interference and REDD1-/- mouse embryonic fibroblasts’, 10.5256/f1000research.7691.d11499121", "appendix": "Author contributions\n\n\n\nLK preformed the studies involving thapsigargin treatment of wild type and knockout REDD1 MEFs and SLR performed the studies using shRNA to knock down REDD1 expression in HEK293 cells. SRK and DLG conceived the study. DLG prepared a first draft of the manuscript. SRK and DLG were responsible for revision of the draft manuscript and all authors have agreed to the final content.\n\n\nCompeting interests\n\n\n\nAt the time this manuscript was initiated DLG was employed by Proteintech. No other known competing interests were present.\n\n\nGrant information\n\nSRK is funded by NIH grants DK15658 and DK094141.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe thank Professor Leif W. Ellisen (Massachusetts General Hospital Cancer Center and Harvard Medical School, Boston, MA, USA) for the provision of and permission to use the REDD1+/+ MEF cell line.\n\n\nReferences\n\nLaplante M, Sabatini DM: mTOR signaling in growth control and disease. Cell. 2012; 149(2): 274–293. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlbert V, Hall MN: mTOR signaling in cellular and organismal energetics. Curr Opin Cell Biol. 2015; 33: 55–66. PubMed Abstract \| Publisher Full Text\n\nBrugarolas J, Lei K, Hurley RL, et al.: Regulation of mTOR function in response to hypoxia by REDD1 and the TSC1/TSC2 tumor suppressor complex. Genes Dev. 2004; 18(23): 2893–2904. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDeYoung MP, Horak P, Sofer A, et al.: Hypoxia regulates TSC1/2-mTOR signaling and tumor suppression through REDD1-mediated 14-3-3 shuttling. Genes Dev. 2008; 22(2): 239–251. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKimball SR, Do AN, Kutzler L, et al.: Rapid turnover of the mTOR complex 1 (mTORC1) repressor REDD1 and activation of mTORC1 signaling following inhibition of protein synthesis. J Biol Chem. 2008; 283(6): 3465–3475. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDennis MD, Coleman CS, Berg A, et al.: REDD1 enhances protein phosphatase 2A-mediated dephosphorylation of Akt to repress mTORC1 signaling. Sci Signal. 2014; 7(335): ra68. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcGhee NK, Jefferson LS, Kimball SR: Elevated corticosterone associated with food deprivation upregulates expression in rat skeletal muscle of the mTORC1 repressor, REDD1. J Nutr. 2009; 139(5): 828–834. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDennis MD, McGhee NK, Jefferson LS, et al.: Regulated in DNA damage and development 1 (REDD1) promotes cell survival during serum deprivation by sustaining repression of signaling through the mechanistic target of rapamycin in complex 1 (mTORC1). Cell Signal. 2013; 25(12): 2709–2716. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMurakami T, Hasegawa K, Yoshinaga M: Rapid induction of REDD1 expression by endurance exercise in rat skeletal muscle. Biochem Biophys Res Commun. 2011; 405(4): 615–619. PubMed Abstract \| Publisher Full Text\n\nEllisen LW, Ramsayer KD, Johannessen CM, et al.: REDD1, a developmentally regulated transcriptional target of p63 and p53, links p63 to regulation of reactive oxygen species. Mol Cell. 2002; 10(5): 995–1005. PubMed Abstract \| Publisher Full Text\n\nShoshani T, Faerman A, Mett I, et al.: Identification of a novel hypoxia-inducible factor 1-responsive gene, RTP801, involved in apoptosis. Mol Cell Biol. 2002; 22(7): 2283–2293. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWhitney ML, Jefferson LS, Kimball SR: ATF4 is necessary and sufficient for ER stress-induced upregulation of REDD1 expression. Biochem Biophys Res Commun. 2009; 379(2): 451–455. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVadysirisack DD, Baenke F, Ory B, et al.: Feedback control of p53 translation by REDD1 and mTORC1 limits the p53-dependent DNA damage response. Mol Cell Biol. 2011; 31(21): 4356–4365. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang H, Kubica N, Ellisen LW, et al.: Dexamethasone represses signaling through the mammalian target of rapamycin in muscle cells by enhancing expression of REDD1. J Biol Chem. 2006; 281(51): 39128–39134. PubMed Abstract \| Publisher Full Text\n\nSofer A, Lei K, Johannessen CM, et al.: Regulation of mTOR and cell growth in response to energy stress by REDD1. Mol Cell Biol. 2005; 25(14): 5834–5845. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChang B, Liu G, Yang G, et al.: REDD1 is required for RAS-mediated transformation of human ovarian epithelial cells. Cell Cycle. 2009; 8(5): 780–786. PubMed Abstract \| Publisher Full Text\n\nDamjanac M, Page G, Ragot S, et al.: PKR, a cognitive decline biomarker, can regulate translation via two consecutive molecular targets p53 and Redd1 in lymphocytes of AD patients. J Cell Mol Med. 2009; 13(8B): 1823–1832. PubMed Abstract \| Publisher Full Text\n\nBritto FA, Begue G, Rossano B, et al.: REDD1 deletion prevents dexamethasone-induced skeletal muscle atrophy. Am J Physiol Endocrinol Metab. 2014; 307(11): E983–E993. PubMed Abstract \| Publisher Full Text\n\nDungan CM, Wright DC, Williamson DL: Lack of REDD1 reduces whole body glucose and insulin tolerance, and impairs skeletal muscle insulin signaling. Biochem Biophys Res Commun. 2014; 453(4): 778–783. PubMed Abstract \| Publisher Full Text\n\nGordon BS, Williamson DL, Lang CH, et al.: Nutrient-induced stimulation of protein synthesis in mouse skeletal muscle is limited by the mTORC1 repressor REDD1. J Nutr. 2015; 145(4): 708–713. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGrainger DL, Kutzler L, Rannels SL, et al.: Dataset 1 in: Validation of a commercially available anti-REDD1 antibody using RNA interference and REDD1-/- mouse embryonic fibroblasts. F1000Research. 2016. Data Source" }	[ { "id": "12690", "date": "08 Mar 2016", "name": "Peter Cavnar", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 titled “Validation of a commercially available anti-REDD1 antibody using RNA interference and REDD1-/- mouse embryonic fibroblasts” shows convincing data that demonstrates the specificity of the commercially available anti-REDD1 rabbit polyclonal antibody from Proteintech, Inc. (Cat. No. 10638-1-AP, lot #00019207). I have some minor concerns that would help strengthen this report if addressed: Concerns:Figure 1 shows data from two replicates using two separate shRNAs targeted to REDD1. This experiment should be replicated at minimum three times. Furthermore, looking at the raw gels can the authors clarify that these two replicates are from two separates sets of HEK cultures and transfections although they were run on a single gel? What are the other lanes in the raw images? Do the authors see any differences in specificity and signal intensities when blocking with BSA as compared to 5% milk? Is the knockdown quantified in Figure 1B statistically significant? Including a protein standard on the gel to emphasize the molecular weight disparity of REDD1 on SDS-PAGE would be helpful. Do the authors observe this migration of 35 kDa compared to the predicted 25 kDa in both the MEFs and HEKs cell lysates? What protein standard (source) was used on these gels that can be observed in the raw data set? Have the authors tried doing a secondary only control? This could help determine if the band they observe in Figure 2 KO cells is being caused by the secondary antibody. Figure 2 says that n=3 however only two gels are shown in the raw image sets. Can the authors include the third gel? Figure 2 raw data set shows two replicates that look like they are from the same blot. Are these from separate cell lysates and Western blots or was were these two replicates completed simultaneously?", "responses": [] }, { "id": "12892", "date": "15 Mar 2016", "name": "Mei Yee Leung", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 Deborah L. Grainger et al, clearly documents the validation of Rabbit anti-Human REDD-1 in a Western blot application. This data has further reinforced the quality of this already well established and cited commercial antibody by demonstrating specificity using RNAi technology and knock out cell lines. The paper has quite conclusively shown that Proteintech Rabbit anti-Human REDD-1, catalogue number 10638-1-AP, Lot #00019207 is an antibody that binds to a specific region of the human REDD-1 full length protein.However, I have the following comments regarding some potential questions this article may generate. Addressing these would mostly likely strengthen this antibody validation paper and allow the reader to better able to make an informed choice when selecting the right anti-REDD-1 antibody.Since the paper addresses only application in Western blot, could this be included in the title? Many tested applications are listed for this antibody, Catalogue No. 10638-1 in the datasheet including IHC, could the validation for this antibody be extended to include ICC? It would be interesting to see whether thapsigargin induced expression of REDD-1 in Western blot is corroborated by the same in ICC. Figure 2. REDD-1 expression in WT- is quite substantial visually. Would you expect this level of expression under physiological 02? Following on from point 3, REDD-1 is only expressed under certain environmental conditions, including hypoxia [reference 10, Ellisen LW et al, 2002], and the latter has been widely used to induce REDD-1 expression in other studies. Some clarification as to why HEK-293 cells cultured in 5%C02 is showing expression of REDD-1 is warranted and should be included in the discussion. Can you explain the choice of using HEK 293 cell line? Also, was there any reason why the other cell lines listed as positive controls in the datasheet wasn’t included? The probability (high or low) of this anti-REDD-1 (DDIT4 -DNA-Damage-Inducible Transcript 4) antibody cross reacting with REDD-2 protein (DDIT4L -DNA-Damage-Inducible Transcript 4-Like) should also discussed. Will there be any consideration for epitope mapping to be implemented in future lots?", "responses": [] } ]	1	https://f1000research.com/articles/5-250
https://f1000research.com/articles/4-1053/v1	12 Oct 15	{ "type": "Research Note", "title": "Effect of the synthesis of rice non-symbiotic hemoglobins 1 and 2 in the recombinant Escherichia coli TB1 growth", "authors": [ "Emma Álvarez-Salgado", "Raúl Arredondo-Peter", "Emma Álvarez-Salgado" ], "abstract": "Non-symbiotic hemoglobins (nsHbs) are widely distributed in land plants, including rice. These proteins are classified into type 1 (nsHbs-1) and type 2. The O2-affinity of nsHbs-1 is very high mostly because of an extremely low O2-dissociation rate constant resulting in that nsHbs-1 apparently do not release O2 after oxygenation. Thus, it is possible that the in vivo function of nsHbs-1 is other than O2-transport. Based on the properties of multiple Hbs it was proposed that nsHbs-1 could play diverse roles in rice organs, however the in vivo activity of rice nsHbs-1 has been poorly analyzed. An in vivo analysis for rice nsHbs-1 is essential to elucidate the biological function(s) of these proteins. Rice Hb1 and Hb2 are nsHbs-1 that have been generated in recombinant Escherichia coli TB1. The rice Hb1 and Hb2 amino acid sequence, tertiary structure and rate and equilibrium constants for the reaction of O2 are highly similar. Thus, it is possible that rice Hb1 and Hb2 function similarly in vivo. As an initial approach to test this hypothesis we analyzed the effect of the synthesis of rice Hb1 and Hb2 in the recombinant E. coli TB1 growth. Effect of the synthesis of the O2-carrying soybean leghemoglobin a, cowpea leghemoglobin II and Vitreoscilla Hb in the recombinant E. coli TB1 growth was also analyzed as an O2-carrier control. Our results showed that synthesis of rice Hb1, rice Hb2, soybean Lba, cowpea LbII and Vitreoscilla Hb inhibits the recombinant E. coli TB1 growth and that growth inhibition was stronger when recombinant E. coli TB1 synthesized rice Hb2 than when synthesized rice Hb1. These results suggested that rice Hb1 and Hb2 could function differently in vivo.", "keywords": [ "Function", "heterologous expression", "in vivo", "Oryza", "oxygen" ], "content": "Introduction\n\nNon-symbiotic hemoglobins (nsHbs) are O2-binding proteins widely distributed in land plants, including rice1. The nsHbs are classified into type 1 and type 2 (nsHbs-1 and nsHbs-2, respectively) based on sequence similarity and O2-affinity2,3. The O2-affinity of nsHbs-1 is very high mostly because of an extremely low O2-dissociation (koff) rate constant3–5 resulting in that nsHbs-1 apparently do not release O2 after oxygenation6. In contrast, the O2-affinity of nsHbs-2 is moderate mostly because of a moderate to high koff rate constant for O2, thus apparently nsHbs-2 easily release O2 after oxygenation2,3,6. Hence, it is possible that the in vivo function of nsHbs-1 is other than O2-transport and that nsHbs-2 function in vivo as O2-carriers.\n\nFive copies (hb1 to 5) of the nshb gene have been detected in the rice genome, which are differentially expressed in embryonic and vegetative organs from plants growing under normal and stress conditions7–10. Based on the available information on the properties of rice nsHbs and data from the analysis of other plant and non-plant Hbs, it was proposed that rice nsHbs could exhibit a variety of functions in vivo, including O2-transport, O2-sensing, NO-scavenging and redox-signaling6,11. However, the in vivo activity of rice nsHbs has been poorly analyzed11. An in vivo analysis for rice nsHbs is essential to elucidate the biological function(s) of these proteins. An approach to analyze the in vivo activity of nsHbs is generating knock out rice for individual nshb genes, however this is complicated because of the existence of five copies of nshb in the rice genome. An alternative approach to analyze the in vivo activity of rice nsHbs is examining individual rice nsHbs in a heterologous system, such as recombinant Escherichia coli. Rice Hb14 and Hb212 are nsHbs-1 that have been generated in recombinant E. coli TB1. The rice Hb1 and Hb2 amino acid sequence4, tertiary structure13 and rate and equilibrium constants for the reaction of O24,12 are highly similar. Thus, it is possible that rice Hb1 and Hb2 function similarly in vivo. As an initial approach to test this hypothesis we analyzed the effect of the synthesis of rice Hb1 and Hb2 in the recombinant E. coli TB1 growth. Our results showed that synthesis of rice Hb1 and Hb2 inhibited the recombinant E. coli TB1 growth and that growth inhibition was stronger when recombinant E. coli TB1 synthesized rice Hb2 than when synthesized rice Hb1.\n\n\nMethods\n\nUntransformed (wild-type) and transformed (recombinant) E. coli TB1 (Invitrogen, CA, USA) containing the constitutive pEMBL18+::Hb14, pEMBL18+::Hb212, pEMBL18+::Lba14, pEMBL18+::LbII15 and pUC18::VHb16 plasmids were grown in LB broth (Sigma-Aldrich, MO, USA) at 37°C with shaking at 200 rpm. Plasmids pEMBL18+::Lba, pEMBL18+::LbII and pUC18::VHb were included as an O2-carrier control since they code for the synthesis of the O2-carrying soybean leghemoglobin a (Lba), cowpea leghemoglobin II (LbII)15,17,18 and Vitreoscilla Hb (VHb)19,20, respectively. The existence of the VHb insert into the pUC18::VHb plasmid was verified by PCR (30 cycles at 55°C/30s for annealing, 72°C/30s for extension and 95°C/30s for denaturation) using specific oligonucleotides (VitHb/ATG: 5´-ATG TTA GAC CAG CAA ACC ATT-3´ and VitHb/TAA: 5´-TTA TTC AAC CGC TTG AGC GTA-3´) designed from the vhb sequence deposited in the Genbank database under the accession number X13516. The existence of the Hb1, Hb2, Lba and LbII inserts into the pEMBL18+::Hb1, pEMBL18+::Hb2, pEMBL18+::Lba and pEMBL18+::LbII plasmids, respectively, was verified by EcoRI- and NcoI (Invitrogen, CA, USA) -double digestion. Inserts were detected by electrophoresis in a 1.4% agarose gel. The existence of recombinant Hbs in cell soluble extracts was verified by SDS-PAGE in a 12.5% polyacrylamide gel. Evaluation of the effect of the Hb synthesis in the recombinant E. coli TB1 growth was performed in 50 ml cultures inoculated with ≈5 × 108 colony forming units from a 20 ml overnight culture. Wild-type E. coli TB1 was included as control. All assays were performed in triplicate. Cell growth was quantitated by spectrophotometry using λ = 650 nm for an 8.5 h period.\n\n\nResults and discussion\n\nElectrophoretic analysis of the PCR reaction and EcoRI- and NcoI-double digestions showed that plasmids isolated from recombinant E. coli TB1 contained inserts corresponding to the rice Hb1, rice Hb2, soybean Lba, cowpea LbII and Vitreoscilla Hb cDNAs (Figure 1A). Likewise, analysis by SDS-PAGE showed that rice Hb1, rice Hb2, soybean Lba, cowpea LbII and Vitreoscilla Hb existed in the soluble extracts of recombinant E. coli TB1 (Figure 1B). This evidence indicated that rice Hb1, rice Hb2, soybean Lba, cowpea LbII and Vitreoscilla Hb were synthesized by recombinant E. coli TB1.\n\n(A) Detection of Vitreoscilla Hb PCR fragment and soybean Lba, cowpea LbII, rice Hb1 and rice Hb2 cDNAs from recombinant E. coli TB1 by agarose gel electrophoresis. PCR fragment and cDNA sizes are within the 435 to 507 base pairs range. Molecular size markers are indicated in base pairs. (B) Detection of Vitreoscilla Hb, soybean Lba, cowpea LbII, rice Hb1 and rice Hb2 proteins (arrow heads) from recombinant E.coli TB1 soluble extracts by SDS-PAGE. A 20 to 50 μg aliquot of total soluble proteins was loaded onto each lane. Recombinant Hb masses are within the 14 to 18.4 KD range. Mass markers are indicated in kD.\n\nFigure 2 shows that synthesis of rice Hb1, rice Hb2, soybean Lba, cowpea LbII and Vitreoscilla Hb inhibited the recombinant E. coli TB1 growth. This was unexpected for soybean Lba, cowpea LbII and Vitreoscilla Hb because these proteins would promote cell growth due to their O2-transport activity15,17–20. However, under the conditions tested in this work apparently soybean Lba, cowpea LbII and Vitreoscilla Hb affected some aspects of the recombinant E. coli TB1 metabolism, possibly owed to the constitutive expression of these proteins into the host cells. Synthesis of rice Hb1 inhibited the recombinant E. coli TB1 growth similarly (∼37%) to the synthesis of soybean Lba, cowpea LbII and Vitreoscilla Hb. This observation suggests that rice Hb1 could function in vivo similarly to O2-carrying Hbs. Likewise, synthesis of rice Hb2 also inhibited the recombinant E. coli TB1 growth. However, growth inhibition was stronger (∼61%) when recombinant E. coli TB1 synthesized rice Hb2 than when synthesized rice Hb1. This observation suggests that rice Hb2 could function in vivo by scavenging O2, possibly owing to its extremely low koff rate constant for O212.\n\nValues (mean ± SD) correspond to three replicates. See the Methods section for experimental details.\n\n\nConclusions\n\nResults presented in this work suggest that in spite of the high similarity between rice Hb1 and Hb2 these proteins could function differently in vivo. In order to elucidate the apparent metabolic effects generated by the synthesis of rice Hb1 and Hb2, future work might focus on the physiological and biochemical characterization of recombinant E. coli TB1. This may include measuring cell respiratory rates and identifying cell proteins and metabolites using oximetry and proteomic and metabolomic approaches, respectively. Results from these analyses could provide valuable information to understand the in vivo function of rice nsHbs.", "appendix": "Author contributions\n\n\n\nEAS and RAP conceived the study. EAS executed the experiments. RAP prepared the first draft of the manuscript. EAS and RAP revised 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 partially financed by SEP-PROMEP (grant number UAEMor-PTC-01-01/PTC23) and Consejo Nacional de Ciencia y Tecnología (CoNaCyT grant numbers 25229N and 42873Q), México, to RA-P.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nAuthors wish to express their gratitude to Dr. Dale A. Webster (Illinois Institute of Technology, USA) for kindly providing the pUC18::VHb plasmid.\n\n\nReferences\n\nGarrocho-Villegas V, Gopalasubramaniam SK, Arredondo-Peter R: Plant hemoglobins: what we know six decades after their discovery. Gene. 2007; 398(1–2): 78–85. PubMed Abstract \| Publisher Full Text\n\nSmagghe BJ, Hoy JA, Percifield R, et al.: Review: correlations between oxygen affinity and sequence classifications of plant hemoglobins. Biopolymers. 2009; 91(12): 1083–1096. PubMed Abstract \| Publisher Full Text\n\nTrevaskis B, Watts RA, Andersson CR, et al.: Two hemoglobin genes in Arabidopsis thaliana: the evolutionary origins of leghemoglobins. Proc Natl Acad Sci USA. 1997; 94(22): 12230–12234. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nArredondo-Peter R, Hargrove MS, Sarath G, et al.: Rice hemoglobins. Gene cloning, analysis and O2-binding kinetics of a recombinant protein synthesized in Escherichia coli. Plant Physiol. 1997; 115(3): 1259–1266. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDuff SM, Wittenberg JB, Hill RD: Expression, purification and properties of recombinant barley (Hordeum sp.) hemoglobin. Optical spectra and reactions with gaseous ligands. J Biol Chem. 1997; 272(27): 16746–16752. PubMed Abstract \| Publisher Full Text\n\nArredondo-Peter R, Hargrove MS, Moran JF, et al.: Plant hemoglobins. Plant Physiol. 1998; 118(4): 1121–1125. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGarrocho-Villegas V, Bustos-Rivera G, Gough J, et al.: Expression and in-silico structural analysis of a rice (Oryza sativa) hemoglobin 5. Plant Physiol Biochem. 2008; 46(10): 855–859. PubMed Abstract \| Publisher Full Text\n\nLira-Ruan V, Ross EJH, Sarath G, et al.: Mapping and analysis of a hemoglobin gene family from rice (Oryza sativa). Plant Physiol Biochem. 2002; 40(3): 199–202. Publisher Full Text\n\nLira-Ruan V, Ruiz-Kubli M, Arredondo-Peter R: Expression of non-symbiotic hemoglobin 1 and 2 genes in rice (Oryza sativa) embryonic organs. Commun Integr Biol. 2011; 4(4): 457–458. PubMed Abstract \| Free Full Text\n\nLira-Ruan V, Sarath G, Klucas RV, et al.: Synthesis of hemoglobins in rice (Oryza sativa var. Jackson) plants growing in normal and stress conditions. Plant Sci. 2001; 161(2): 279–287. PubMed Abstract \| Publisher Full Text\n\nArredondo-Peter R, Moran JF, Sarath G: Rice (Oryza) hemoglobins [version 2; referees: 2 approved]. F1000Res. 2014; 3: 253. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmagghe BJ, Sarath G, Ross E, et al.: Slow ligand binding kinetics dominate ferrous hexacoordinate hemoglobin reactivities and reveal differences between plants and other species. Biochemistry. 2006; 45(2): 561–570. PubMed Abstract \| Publisher Full Text\n\nGopalasubramaniam SK, Garrocho-Villegas V, Rivera GB, et al.: Use of in silico (computer) methods to predict and analyze the tertiary structure of plant hemoglobins. Meth Enzymol. 2008; 436: 393–410. PubMed Abstract \| Publisher Full Text\n\nHargrove MS, Barry JK, Brucker EA, et al.: Characterization of recombinant soybean leghemoglobin a and apolar distal histidine mutants. J Mol Biol. 1997; 266(5): 1032–1042. PubMed Abstract \| Publisher Full Text\n\nArredondo-Peter R, Moran JF, Sarath G, et al.: Molecular cloning of the cowpea leghemoglobin II gene and expression of its cDNA in Escherichia coli. Purification and characterization of the recombinant protein. Plant Physiol. 1997; 114(2): 493–500. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWakabayashi S, Matsubara H, Webster DA: Primary sequence of a dimeric bacterial haemoglobin from Vitreoscilla. Nature. 1986; 322(6078): 481–483. PubMed Abstract \| Publisher Full Text\n\nAppleby CA: The origin and functions of haemoglobin in plants. Sci Progress. 1992; 76(3/4): 365–398. Reference Source\n\nAppleby CA, Bradbury JH, Morris RJ, et al.: Leghemoglobin. Kinetic, nuclear magnetic resonance, and optical studies of pH dependence of oxygen and carbon monoxide binding. J Biol Chem. 1983; 258(4): 2254–2259. PubMed Abstract\n\nChi P, Webster DA, Stark BC: Vitreoscilla hemoglobin aids respiration under hypoxic conditions in its native host. Microbiol Res. 2009; 164(3): 267–275. PubMed Abstract \| Publisher Full Text\n\nDikshit RP, Dikshit KL, Liu YX, et al.: The bacterial hemoglobin from Vitreoscilla can support the aerobic growth of Escherichia coli lacking terminal oxidases. Arch Biochem Biophys. 1992; 293(2): 241–245. PubMed Abstract \| Publisher Full Text" }	[ { "id": "11632", "date": "06 Jan 2016", "name": "Robert Hill", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 approach to possibly differentiating between the functions of this group of proteins. A fundamental question that arises out of this work is why do hemoglobin proteins inhibit E. coli growth? The growth differences between the rice Pgb1.1-transformed line and the rice Pgb1.2 line is particularly interesting. I would be cautious, however, in attempting to interpret the results with respect to the proteins possibly behaving as oxygen carriers/transporters. My concerns are based on the following:Why would a unicellular organism without mitochondria require an oxygen carrier since oxidative phosphorylation occurs on the plasma membrane? If plant phytoglobins have an oxygen carrier function would you not expect the two class 1 phytoglobins to have the same effect since they both have similar oxygen binding characteristics? Why would you anticipate that Pgb1.2 might participate more in NO scavenging than Pgb1.1 in E. coli if they both have similar configurations in the heme pocket? Is it not the class 2 Pgbs that are suggested to possibly being less amenable to NO scavenging? Is it possible that the expression of the class 2 protein is interfering with some function of the native flavohemoglobin in E. coli? Although E. coli does not have a true nucleus, is it possible that the protein is specifically interfering with transcriptional/translation functions in the chromosome, e.g., the N-end rule pathway as one possibility?My other comment concerns the terminology. The individuals who work in this area agreed to forego the use of the term \"nonsymbiotic hemoglobins\" at an international meeting in 2014, replacing it with \"phytoglobin\", since the original designation does not appropriately describe the protein. I would hope that the authors consider modifying the manuscript to ensure that the name change becomes recognized in the literature.", "responses": [ { "c_id": "1752", "date": "08 Jan 2016", "name": "Raul Arredondo-Peter", "role": "Author Response", "response": "We thank Dr. Robert Hill for evaluating this article and providing constructive comments and suggestions.We agree with Dr. Hill´s comments corresponding to questions 1 to 5. Undoubtedly they should also be considered in future research focused to elucidate the physiological effects of the synthesis of rice non-symbiotic hemoglobins 1 and 2 in recombinant E. coli TB1.Regarding the terminology, we decided to not change the term “non-symbiotic hemoglobins 1 and 2” by “phytoglobins 1.1 and 1.2” (which was accepted in the 2014 XVIII Oxygen-Binding and Sensing Proteins meeting) because details for the accepted nomenclature have not been published. Thus, the accepted nomenclature is not yet widely available to individuals working/interested in the plant hemoglobins field. Hence, replacing the term non-symbiotic hemoglobins 1 and 2 (which has been used for many years in the literature) by the novel term phytoglobins 1.1 and 1.2 could result as confusing to readers of this article." } ] }, { "id": "12624", "date": "23 Feb 2016", "name": "Angel Matilla", "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 work by Álvarez-Salgado and Arredondo-Peter (2015) was carefully evaluated. Broadly, this research is worthy of achieving an approval in F1000Research. However, before it achieves this status, is necessary to carry out some minor modifications.Thus, the first paragraph of the Introduction, referred to the different affinity of nsHbs1 and nsHb2 for O2, needs to include some recent references (eg. Hoy and Hargrove, 2008; Smagghe et al, 2009; Thiel et al., 2011; among others). Likewise, in the second paragraph of the Introduction (ie. Based on the available information…. NO scavenging and redox-signaling) the following latest references must also be added (ie. Siddiqui et al., 2010; Vigeolas et al., 2010, among others).With respect to Res & Discuss, (i) the first paragraph should include some reference to show that the bands referred by the authors (Fig. 1A) specifically belong to rice (Hb1, Hb2), soybean (LBA), cowpea (LbII) and Vitreoscilla (Hb) cDNAs. This fact is key in this work. Likewise, bands corresponding to VHb and Lba (Fig. 1B) are confusing to the reader; (ii) I would eliminate from Fig. 2 the results of LBA, LbII and HBv growth (include as data not shown) because the main importance of this work are the results concerning nb1 and nbII; (iii) \"... these proteins would promote cell growth due to their O2-transport activity\"; this conclusion is based in old results and is very risky; this growth promotion should also be referred to higher plants?; please discuss; and (iv) I repeat, some actual references must be also included into discussion.", "responses": [] } ]	1	https://f1000research.com/articles/4-1053
https://f1000research.com/articles/5-240/v1	29 Feb 16	{ "type": "Review", "title": "Genetics and Therapeutics in Pediatric Ulcerative Colitis: the Past, Present and Future", "authors": [ "Luis Sifuentes-Dominguez", "Ashish S. Patel", "Luis Sifuentes-Dominguez" ], "abstract": "Ulcerative colitis (UC) is a relapsing and remitting disease with significant phenotypic and genotypic variability. Though more common in adults, UC is being increasingly diagnosed in childhood. The subsequent lifelong course of disease results in challenges for the patient and physician. Currently, there is no medical cure for UC. Even though surgical removal of the colon can be curative, complications including infertility in females make colectomy an option often considered only when the disease presents with life-threatening complications or when medical management fails. One of the greatest challenges the clinician faces in the care of patients with UC is the inability to predict at diagnosis which patient is going to respond to a specific therapy or will eventually require surgery. This therapeutic conundrum frames the discussion to follow, specifically the concept of individualized or personalized treatment strategies based on genetic risk factors. As we move to therapeutics, we will elucidate traditional approaches and discuss known and novel agents. As we look to the future, we can expect increasing integrated approaches using several scientific disciplines to inform how genetic interactions shape and mold the pathogenesis and therapeutics of UC.", "keywords": [ "Ulcerative colitis", "genetic risk factors", "therapeutics", "personalized treatment strategies" ], "content": "\n\nUlcerative colitis (UC) is a chronic relapsing and remitting disease characterized by inflammation, ulceration, and bleeding in the large intestine. UC can present with significant phenotypic and genetic variability. This variability results in substantial difficulty as it relates to the appropriate therapeutic approach for an individual patient. We will first investigate the concept of individualized or personalized treatment strategies by describing the current understanding of genetics in pediatric UC. We will then move to the therapeutic discussion, focusing on traditional approaches, known agents in new roles, and novel agents and targets. Our intent will be to frame the therapeutic discoveries of the past, present, and future on the timeline of genetic discovery.\n\n\nGenetics of pediatric ulcerative colitis\n\nEvolving technologies are allowing us to dissect the genetic architecture of inflammatory bowel disease (IBD) (Figure 1). Early epidemiological observations served as the basis for a genetic model of IBD, and a growing body of evidence continues to support and strengthen this notion. Genetic discoveries over the past century have greatly advanced our understanding of UC pathogenesis. These discoveries have provided great insight into disease-associated pathways and have had a dramatic influence over drug discovery and development.\n\nInterestingly, Crohn’s disease (CD) and UC share a large number of the described susceptibility loci, suggesting that they may share a common genetic background. Clinical differences, however, would suggest that different genes and pathways are involved in each disease and thus these differences may explain the discrepant phenotypes and may certainly provide the basis for targeted drug development.\n\n\nGenetic epidemiology\n\nClinical recognition of disease clustering in select families has long been appreciated1,2. Estimated IBD prevalence among family members of patients with UC is 7.9% to 12%3,4, with high concordance for disease type. Although these observations may be explained by shared environmental factors in family members, further proof of genetic involvement has come from twin studies that show higher concordance rates of IBD among monozygotic twins. A recent analysis of available twin studies showed a 15.4% concordance rate for UC among monozygotic twins compared to 3.9% in dizygotic counterparts5. Yet UC does not exhibit traditional Mendelian inheritance and thus is a complex polygenic disease.\n\nIBD clusters not only in families but also among populations. IBD prevalence is significantly higher in whites than in non-whites, notably in those of European ancestry6. Ethnic aggregation is particularly seen in Ashkenazi Jews in whom disease prevalence is highest7,8. On the other hand, disease incidence continues to increase worldwide9, particularly in westernized societies, enforcing the concept that gene-environment interactions are at the core of IBD pathogenesis.\n\n\nLinkage studies\n\nIn the latter part of the 20th century, association and linkage disequilibrium studies yielded fruitful evidence of loci associated with IBD susceptibility. The first IBD susceptibility locus was described in 199610 and mapped to chromosome 16, where NOD2 would later be identified as a candidate CD-associated gene11,12. Similar studies followed and genetic heterogeneity of both CD and UC was quickly appreciated. It became apparent that many genes may also participate in the phenotypic expression of UC.\n\nTo overcome the limitations of narrow genome coverage of linkage disequilibrium studies in the analysis of polygenic diseases, several key developments would have to occur. As pointed out by Mathew13, the creation of dense single nucleotide polymorphism (SNP) libraries, the development of a high-resolution genetic map that allowed measurement of association between SNPs (HapMap), and the advent of high-throughput genotyping methods provided the necessary tools for further discovery of IBD susceptibility loci through genome-wide association studies (GWAS).\n\n\nThe Genome-Wide Association Study era\n\nTo study polygenic diseases, GWAS rely on the premise that genome-wide genetic variations that may be associated with a specific disease will occur with greater frequency in subjects with the disease when compared to healthy controls. These associations must withstand rigorous statistical methods. Because signals from individual loci may be very small, GWAS require large patient and control cohorts to establish significance.\n\nMore than any other complex polygenic disease, IBD has been the subject of numerous GWAS and meta-analyses14–24. Results from these studies have helped unravel the inner workings of IBD pathophysiology and numerous genes affecting key pathways have been described (relevant pathways discussed below). More recently, meta-analyses combining GWAS and Immunochip data (genotyping chip containing >196,000 polymorphisms)25 have dramatically increased the number of IBD-associated loci.\n\nThe first publication using this strategy combined data from 75,000 samples26. This study expanded the known IBD susceptibility loci to 163, with 23 of them UC specific and 110 (67%) shared by both CD and UC. Three important observations were also made by this study. First, IBD shows overlap with other immune-related diseases such as primary immunodeficiencies, spondyloarthropathies, and psoriasis, suggesting a common origin and pathophysiological basis. Second, IBD loci were significantly enriched in genes implicated in susceptibility to mycobacterial diseases, perhaps conserved by evolutionary pressure. Third, a large proportion of loci were found in non-coding regions, adding fuel to the idea that non-coding variation may be responsible for differential expression of complex traits.\n\nA follow-up study using a similar approach incorporated more than 96,000 samples from European, East Asian, Indian, and Iranian populations, increasing the number of susceptibility loci to 20027. Of notable interest was the finding that most loci were shared by all populations studied, implying that causal variants are common, even in ethnically diverse individuals. Admixed populations, such as African-Americans, have also recently been scrutinized using the Immunochip, and previously established loci were replicated in this population as well28.\n\n\nCurrent state of ulcerative colitis genetics\n\nA common finding among GWAS and meta-analyses is increased signal enrichment in regions that participate in intestinal immune homeostasis. Functional characterization of genes found in these loci has led to important discoveries in the pathways involved in disease. Below, we describe relevant genes implicated in pathways associated with UC.\n\nBecause of their roles in antigen presentation and T-cell priming, HLA gene variants have long been considered prime candidates for IBD susceptibility29,30. Indeed, pre-GWAS era linkage disequilibrium studies first described genetic association of IBD susceptibility to major histocompatibility complex (MHC) regions. Hampered by difficulties inherent to the linkage disequilibrium architecture of the area, susceptibility loci identification was slow31. However, all GWAS to date have continued to report a strong association between HLA loci and UC. A recent study performed high-density mapping of the MHC region in over 32,000 patients with IBD32. This study found significant enrichment in MHC class II alleles: HLA DRB1, HLA DQA1, and HLA-DRB101:03 in particular were strongly associated with UC. The HLA-DRB101:03 allele is of particular interest as it also showed a strong association with CD colitis and other studies have linked it to pancolitic phenotype in UC33. Goyette also found decreased class II HLA heterozygosity, suggesting that broader colonic antigen recognition is important in protection against UC development.\n\nThese associations are especially intriguing for pediatric IBD, since young patients, in particular those less than 8 years of age, are more prone to developing extensive colonic disease34. Two pediatric IBD GWAS studies have replicated findings of HLA polymorphisms with UC susceptibility17,18.\n\nImmune tolerance to food and the normal intestinal microbiota is crucial for the control of intestinal homeostasis. A critical element in maintaining intestinal immune homeostasis is epithelial integrity. In addition, the observation that MUC2-/- deficient mice develop spontaneous colitis35 suggests that the intestinal mucus layer contributes to barrier function and colonic immune homeostasis. Epithelial barrier function in UC is impaired36 and active UC is characterized by mucin depletion.\n\nNot surprisingly, polymorphisms associated with UC have been found in genes controlling key aspects of barrier function. These include polymorphisms in ECM1, believed to control epithelial basement membrane integrity37,38. SNPs in the CDH1 locus, encoding the adherens junction protein E-cadherin required for tight junction formation, HNF4a, responsible for epithelial differentiation, and LAMB1, encoding the β1 subunit of basement membrane laminin, have also been identified14 and replicated39. Finally, polymorphisms in GNA12, associated with tight junction assembly via interactions with Zo-1 and Src, have recently been described40.\n\nThe unfolded protein response (UPR) is a stress response of the endoplasmic reticulum (ER) aimed at maintaining cellular homeostasis, especially in highly secretory cells such as Paneth cells and goblet cells. ER stress has been linked to environmental influences and loss or dysregulation of the UPR leads to activation of apoptotic pathways. The intestinal surface is the largest epithelium in contact with the environment, so it is not surprising that genes controlling the UPR pathway have been associated with IBD pathogenesis. Bertolotti et al. reported that mice lacking IRE1β expression, a component of the UPR, develop exacerbated dextran sulfate sodium (DSS)-induced colitis41. Human UPR-associated polymorphisms have also been described. Kaser et al. reported associations of XBP1 variants, a transcription factor involved in the UPR, with development of UC42. Similarly, susceptibility-associated variants in PTPN2, a protein tyrosine phosphatase linked to UPR and autophagosome formation43,44, have also been described40,45.\n\nThe interleukin (IL)-23 pathway plays a key role in expansion and differentiation of T helper (Th) and innate immune cells. Abnormalities in the IL-23 signaling pathway have been linked to development of UC in several cohorts23. Other components of the pathway such as JAK2 and TYK2 have also been linked to disease pathogenesis37. The importance of this pathway in regulating pro-inflammatory effects seen in IBD is highlighted by the clinical improvement seen in patients with CD when treated with ustekinumab, a monoclonal antibody directed against the p40 subunit shared by IL-12 and IL-2346. The ongoing phase 3 international multicenter study UNIFI aims to evaluate this monoclonal antibody in maintenance and induction of patients with moderate to severe UC47.\n\nOther Th17-centric polymorphisms have been associated with UC, including those in NFKBIZ, a regulator of Th17 cell development, and AHR, a transcription factor involved in Th17 cytokine expansion27. Additional important polymorphisms in blocks containing immune regulatory genes have been described. For a detailed review please refer to the article by Khor et al.48.\n\n\nGenome-Wide Association Study limitations and missing heritability\n\nResults from meta-analyses employing Immunochip indicate that the expected UC disease variance from susceptibility loci is only 8.2%. Therefore, there is a wide gap in the expected disease heritability, a problem termed “missing heritability”. One approach to tackling the missing heritability problem in IBD is the study of patients with single gene disorders with large effects. These monogenic forms of IBD are highly penetrant and cluster in young children, the very early onset IBD group (VEOIBD). Indeed, numerous single gene disorders have been recently described and include mutations in ADAM17, NCF2, NCF4, TTC7A, IL-10, and XIAP49–54.\n\nAlthough GWAS have dramatically influenced our understanding of complex diseases, one limitation of such studies is that they neglect contribution from rare variants, those with minor allele frequencies less than 5%. High effect rare risk variants missed by GWAS may be contained within known IBD susceptibility loci. Two studies have sought to identify causal functionally relevant genetic variants contained within IBD susceptibility loci by employing deep next-generation sequencing. Results from these studies pointed to three new mutations associated with UC. While variants in CARD9 and IL23R were protective, a variant in RNF186, a RING ubiquitin ligase, was found to confer risk55,56. In addition to reporting novel associations with IBD risk, these studies provide new insight into future methodological strategies for gene discovery in IBD.\n\nAdapted from Expert Rev Gastroenterol Hepatol. 2009 513–34.\n\n\nImplications for translation\n\nOne of the most promising aspects of genetics is the use of genetic markers to help inform clinical decision-making. In this regard, IBD genetics has made significant strides.\n\nCurrently, pediatric UC diagnosis is based on aggregate clinical, endoscopic, imaging, and histopathological findings as suggested by North American and European working groups57,58. The rapid pace of discovery of UC-associated genetic variants suggests that genetic diagnosis is quickly unfolding. To date, UC-specific diagnostic approaches utilizing genetic expression analysis from colonic biopsies59,60, composite genetic risk scores61, and combined serological and genetic markers62 have shown promise, yet application of these tools has yet to invade day-to-day clinical practice. The VEOIBD age group is an exception. As monogenic forms of IBD cluster in this age group, exome and targeted gene chips are increasingly being suggested and used in the genetic diagnosis of VEOIBD63,64.\n\nThe best-described genotype-phenotype associations in IBD are NOD2 variants and ileal CD65–67. Recent evidence has also pointed to UC-associated polymorphisms and phenotype associations. MHC-associated variants are of notable interest. As mentioned earlier, polymorphisms in the MHC region have been associated with extensive colonic involvement and colectomy33,68. Recently, the international IBD Genetics Consortium (IIBDGC) reported the results of the largest genotype-phenotype study to date, incorporating data from more than 34,000 IBD patients. Results pointed to three loci associated with disease subphenotype: NOD2, MHC, and 3p21. MHC loci were again associated with extensive colonic involvement69.\n\nThe ability to predict disease course at diagnosis as well as early identification of patients who will develop severe and medically refractory UC may result in changes in treatment algorithms.\n\nHaritunians et al. developed a risk score utilizing a GWAS approach in a well-characterized cohort of patients with medically refractory UC. A modeled risk score using 46 SNPs explained 48% of the variance for colectomy risk in the population studied, with a sensitivity of 79% and a specificity of 86%. Further, this study confirmed associations of severe UC with SNPs within the MHC region, as well as SNPs in TNFSF15, IL-10, IL-12B, 12q15, ZPF90, KIF1A, and GSDML/ORMDL370. A study using the same strategy incorporated data from 703 UC patients from 40 sites within the IIBDGC. This study replicated an association between rs2403456 (11p15.3) with medically refractory, severe UC71.\n\nIn order to identify genetic predictors of response to biologic therapy, Arijs et al. studied colonic mucosal gene expression signatures from two cohorts of anti-tumor necrosis factor (anti-TNF)-naïve UC patients receiving biologic induction with infliximab. Microarray-based colonic biopsy profiling was used to develop a prediction response probe set. Five genes − osteoprotegerin, stanniocalcin-1, prostaglandin-endoperoxide synthase 2, IL-13 receptor alpha-2, and IL-11 − were found to be differentially expressed in responders versus non-responders. The genes identified are involved in adaptive immune response signaling and TNF pathways72. Of note, osteoprotegerin has been suggested as a stool marker for diagnosis and assessment of treatment response in pediatric UC73,74.\n\nThe intent of our discussion previously on genetics and UC is seeded in the hope that personalization of therapies based on genetics will change disease outcomes and potentially lead us to a cure of UC in the future. Though corticosteroids and mesalamine remain the mainstays of current therapy, the following sections will outline other options within our armamentarium with a special focus on the resurgence of drugs like methotrexate, the debate around antibiotics, and a brief look at potential newcomers.\n\nMethotrexate, an immunosuppressant developed in 1948, has been widely used initially in leukemia and now in oncologic, rheumatologic, and autoimmune diseases. Methotrexate inhibits lymphocyte proliferation as a folate antagonist75. Methotrexate has recently garnered attention as an alternative oral or subcutaneous agent in the treatment of UC, particularly in patients who have failed or become intolerant to 6-mercaptopurine (6-MP) or infliximab therapy. Two Cochrane reviews from 2014 and 2015, assessing induction and remission success, respectively, showed no significant difference between methotrexate and placebo in patients with UC76,77. Currently there are two large prospective trials evaluating the potential of methotrexate in UC. The Comparison of Methotrexate versus Placebo in Corticosteroid-dependent UC (METEOR) trial has completed enrollment and is closed. The Methotrexate Response In the Treatment of UC (MERIT-UC) trial is currently recruiting as multicenter prospective trials comparing methotrexate to placebo in patients who are steroid dependent or who failed 6-MP or infliximab therapy.\n\nAntibiotics present an interesting clinical discussion in the setting of UC. Dysbiosis is present in patients with IBD, even at diagnosis. Previous work has shown that in patients with acute severe colitis, those with a more diverse microbiome at diagnosis respond better to steroid therapy versus those with a less diverse microbiome needing salvage medical therapy78. A recent paper by Turner et al. described the use of an oral broad-spectrum antibiotic cocktail (metronidazole, amoxicillin, doxycycline, and vancomycin [MADoV]) in pediatric UC patients who had failed other traditional therapies (steroids, oral immunosuppressants including azathioprine and tacrolimus, and anti-TNF agents, both infliximab and adalimumab). A total of 7 out of 15 patients achieved complete short-term remission with the antibiotic cocktail. A larger pediatric randomized controlled trial to assess this intervention is underway79. Additionally, in the discussion of dysbiosis, fecal microbial transplantation (FMT) has been proposed in the treatment of UC. There are several active recruiting studies ongoing; however, the only published data on the subject by Kunde et al. suggested a promising response in a pilot study of 10 children over 1 month80.\n\nThe latest treatment for UC is a humanized monoclonal antibody named vedolizumab. An α4β7 integrin blocker, the medication targets inflammation by blocking the recruitment of lymphocytes specifically to the gut without interfering with trafficking to the central nervous system. This gut-specific feature makes it unique to the current group of therapeutics by isolating the affected area of involvement and subsequently decreasing the systemic effects81.\n\nNovel therapeutic targets continue to be developed and there are multiple drugs in different phases of development (Figure 2).\n\nNew therapeutic agents for ulcerative colitis with drug targets in parenthesis47,83–90.\n\nTreatment algorithms continue to evolve in UC and are increasingly utilizing predictive models to determine the best potential therapies for an individual patient. An integral part of the predictive model will be our continued advancement of the understanding of genetic data. Additionally, clinical variables have been recognized to impact the determination of initial therapy and as a predictive tool for choosing a more aggressive therapy in patients at risk for a poor outcome. Schechter et al. described the importance of the PUCAI score at baseline and moreover at the 3-month mark. The 3-month PUCAI was most predictive of sustained response and steroid-free remission at 1 year. Several other variables, including elevated C-reactive protein (CRP), hypoalbuminemia, and anemia have also been described in other studies, though not found to be significant in the Schechter model82.\n\nAlthough a cure for polygenic complex diseases is still a hope, lessons from VEOIBD provide proof of principle that, indeed, certain well-characterized monogenic forms of IBD may be amenable to cure. Glocker et al. described a series of patients with infantile fistulizing enterocolitis resulting from mutations in IL-10RA and IL-10RB. Allogeneic stem cell transplantation in one of the patients resulted in complete resolution of gastrointestinal symptoms50. Similarly, Worthey et al. described a patient with onset of fistulizing colonic disease at 15 months of age. Exome sequencing of this patient pointed to hemizygous missense mutation affecting XIAP; cord blood hematopoietic stem cell transplantation led to disease remission52.\n\n\nConclusions\n\nThe future of UC genetics looks bright, but there are a few hurdles to overcome. Current GWAS/Immunochip approaches are likely to have exhausted loci discovery in European ancestry populations; further multi-ethnic studies might lead to fruitful discoveries. Fine mapping and next-generation sequencing will provide additional tools for IBD-associated gene discoveries and may help explain some of the missing heritability in IBD, as well as provide a basis for functional studies that will in turn pave the way for targeted drug development.\n\nLastly, it is important to consider how genetics will influence daily clinical practice in the years to come and how clinicians will choose to adopt newer technologies. Certainly, the large amount of data associated with genetic information may be daunting to the general gastroenterology practitioner, and it is the job of researchers, IBD specialists, and working guideline groups to provide tools for daily clinical use.\n\nAs we look to the future, we can expect increasing integrated approaches using several biological disciplines to inform how genetic interactions shape and mold the pathogenesis and therapeutics of UC.", "appendix": "Competing interests\n\n\n\nThe authors declare that they 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\nAllchin WH: A Discussion on “Ulcerative Colitis.”: Introductory Address. Med Sect. 1909; 2(Med Sect): 59–75. PubMed Abstract \| Free Full Text\n\nKirsner JB, Spencer JA: Family Occurrences of Ulcerative Colitis, Regional Enteritis, and Ileocolitis. Ann Intern Med. 1963; 59(2): 133–144. 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PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nBeaudoin M, Goyette P, Boucher G, et al.: Deep resequencing of GWAS loci identifies rare variants in CARD9, IL23R and RNF186 that are associated with ulcerative colitis. PLoS Genet. 2013; 9(9): e1003723. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nRivas MA, Beaudoin M, Gardet A, et al.: Deep resequencing of GWAS loci identifies independent rare variants associated with inflammatory bowel disease. Nat Genet. 2011; 43(11): 1066–1073. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nLevine A, Koletzko S, Turner D, et al.: ESPGHAN revised porto criteria for the diagnosis of inflammatory bowel disease in children and adolescents. J Pediatr Gastroenterol Nutr. 2014; 58(6): 795–806. PubMed Abstract\n\nNorth American Society for Pediatric Gastroenterology, Hepatology, and Nutrition; Colitis Foundation of America, Bousvaros A, et al.: Differentiating ulcerative colitis from Crohn disease in children and young adults: report of a working group of the North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition and the Crohn’s and Colitis Foundation of America. J Pediatr Gastroenterol Nutr. 2007; 44(5): 653–674. PubMed Abstract \| Publisher Full Text\n\nvon Stein P, Lofberg R, Kuznetsov NV, et al.: Multigene analysis can discriminate between ulcerative colitis, Crohn’s disease, and irritable bowel syndrome. Gastroenterology. 2008; 134(7): 1869–81; quiz 2153–4. PubMed Abstract \| Publisher Full Text\n\nJanczewska I, Kapraali M, Saboonchi F, et al.: Clinical application of the multigene analysis test in discriminating between ulcerative colitis and Crohn’s disease: a retrospective study. Scand J Gastroenterol. 2012; 47(2): 162–169. PubMed Abstract \| Publisher Full Text\n\nAnanthakrishnan AN, Huang H, Nguyen DD, et al.: Differential effect of genetic burden on disease phenotypes in Crohn’s disease and ulcerative colitis: analysis of a North American cohort. Am J Gastroenterol. 2014; 109(3): 395–400. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPlevy S, Silverberg MS, Lockton S, et al.: Combined serological, genetic, and inflammatory markers differentiate non-IBD, Crohn’s disease, and ulcerative colitis patients. Inflamm Bowel Dis. 2013; 19(6): 1139–1148. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKammermeier J, Drury S, James CT, et al.: Targeted gene panel sequencing in children with very early onset inflammatory bowel disease--evaluation and prospective analysis. J Med Genet. 2014; 51(11): 748–755. PubMed Abstract \| Publisher Full Text\n\nUhlig HH, Schwerd T, Koletzko S, et al.: The diagnostic approach to monogenic very early onset inflammatory bowel disease. Gastroenterology. 2014; 147(5): 990–1007.e3. PubMed Abstract \| Publisher Full Text\n\nAbreu MT, Taylor KD, Lin YC, et al.: Mutations in NOD2 are associated with fibrostenosing disease in patients with Crohn’s disease. Gastroenterology. 2002; 123(3): 679–688. PubMed Abstract \| Publisher Full Text\n\nBrant SR, Picco MF, Achkar JP, et al.: Defining complex contributions of NOD2/CARD15 gene mutations, age at onset, and tobacco use on Crohn’s disease phenotypes. Inflamm Bowel Dis. 2003; 9(5): 281–289. PubMed Abstract \| Publisher Full Text\n\nNewman B, Silverberg MS, Gu X, et al.: CARD15 and HLA DRB1 alleles influence susceptibility and disease localization in Crohn’s disease. Am J Gastroenterol. 2004; 99(2): 306–315. PubMed Abstract \| Publisher Full Text\n\nde la Concha EG, Fernandez-Arquero M, Lopez-Nava G, et al.: Susceptibility to severe ulcerative colitis is associated with polymorphism in the central MHC gene IKBL. Gastroenterology. 2000; 119(6): 1491–1495. PubMed Abstract \| Publisher Full Text\n\nCleynen I, Boucher G, Jostins L, et al.: Inherited determinants of Crohn’s disease and ulcerative colitis phenotypes: a genetic association study. Lancet. 2016; 387(10014): 156–67. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nHaritunians T, Taylor KD, Targan SR, et al.: Genetic predictors of medically refractory ulcerative colitis. Inflamm Bowel Dis. 2010; 16(11): 1830–1840. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nRadford-Smith G, Doecke JD, Lees CW, et al.: Su1762 Clinical and Molecular Characterization of Medically Refractory Acute, Severe Colitis: Preliminary Results From the International Inflammatory Bowel Disease Genetics Consortium (IIBDGC) Immunochip Study. Gastroenterology. 2015; 144(5, Supplement 1): S-470. Publisher Full Text\n\nArijs I, Li K, Toedter G, et al.: Mucosal gene signatures to predict response to infliximab in patients with ulcerative colitis. Gut. 2009; 58(12): 1612–1619. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nSylvester FA, Turner D, Draghi A 2nd, et al.: Fecal osteoprotegerin may guide the introduction of second-line therapy in hospitalized children with ulcerative colitis. Inflamm Bowel Dis. 2011; 17(8): 1726–1730. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nSkinner A, Lerer T, Wyzga N, et al.: S1225 Fecal Osteoprotegerin: A Marker for Pediatric Ulcerative Colitis At Diagnosis - a Pilot Study. Gastroenterology. 2008; 134(4, Supplement 1): A-205. Publisher Full Text \| Faculty Opinions Recommendation\n\nHerfarth HH, Osterman MT, Isaacs KL, et al.: Efficacy of methotrexate in ulcerative colitis: failure or promise. Inflamm Bowel Dis. 2010; 16(8): 1421–1430. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChande N, MacDonald JK, McDonald JW: Methotrexate for induction of remission in ulcerative colitis. Cochrane Database Syst Rev. 2007; (4): CD006618. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nWang Y, MacDonald JK, Vandermeer B, et al.: Methotrexate for maintenance of remission in ulcerative colitis. Cochrane Database Syst Rev. 2015; 8: CD007560. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nMichail S, Durbin M, Turner D, et al.: Alterations in the gut microbiome of children with severe ulcerative colitis. Inflamm Bowel Dis. 2012; 18(10): 1799–1808. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nTurner D, Levine A, Kolho KL, et al.: Combination of oral antibiotics may be effective in severe pediatric ulcerative colitis: a preliminary report. J Crohns Colitis. 2014; 8(11): 1464–1470. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nKunde S, Pham A, Bonczyk S, et al.: Safety, tolerability, and clinical response after fecal transplantation in children and young adults with ulcerative colitis. J Pediatr Gastroenterol Nutr. 2013; 56(6): 597–601. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nFeagan BG, Rutgeerts P, Sands BE, et al.: Vedolizumab as induction and maintenance therapy for ulcerative colitis. N Engl J Med. 2013; 369(8): 699–710. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nSchechter A, Griffiths C, Gana JC, et al.: Early endoscopic, laboratory and clinical predictors of poor disease course in paediatric ulcerative colitis. Gut. 2015; 64(4): 580–588. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nSnapper SB: Low Dose IL-2 for Ulcerative Colitis. In: ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US), NLM Identifier: NCT02200445. 2000. Reference Source\n\nKyowa Hakko Kirin Pharma, Inc: Study of a Monoclonal Antibody KHK4083 in Moderate Ulcerative Colitis. In: ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US), NLM Identifier: NCT02647866. 2000. Reference Source\n\nMayer L, Sandborn WJ, Stepanov Y, et al.: Anti-IP-10 antibody (BMS-936557) for ulcerative colitis: a phase II randomised study. Gut. 2014; 63(3): 442–450. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPfizer: Long-Term Safety Of PF-00547659 In Ulcerative Colitis (TURANDOT II). In: ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US), NLM Identifier: NCT01771809. 2000. Reference Source\n\nCelgene Corporation: Efficacy and Safety Study of Apremilast to Treat Active Ulcerative Colitis (UC). In: ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US), NLM Identifier: NCT02289417. 2000. Reference Source\n\nDanese S, Rudziński J, Brandt W, et al.: Tralokinumab for moderate-to-severe UC: a randomised, double-blind, placebo-controlled, phase IIa study. Gut. 2015; 64(2): 243–249. PubMed Abstract \| Publisher Full Text\n\nHerrlinger KR, Diculescu M, Fellermann K, et al.: Efficacy, safety and tolerability of vidofludimus in patients with inflammatory bowel disease: the ENTRANCE study. J Crohns Colitis. 2013; 7(8): 636–643. PubMed Abstract \| Publisher Full Text\n\nPfizer: Long-Term Study Of CP-690,550 In Subjects With Ulcerative Colitis (OCTAVE). In: ClinicalTrials.gov [Internet]. Bethesda (MD): National Library of Medicine (US), NLM Identifier: NCT01470612. 2000. Reference Source" }	[ { "id": "12671", "date": "29 Feb 2016", "name": "Jeffrey Hyams", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12672", "date": "29 Feb 2016", "name": "Francisco Sylvester", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-240
https://f1000research.com/articles/4-1521/v1	30 Dec 15	{ "type": "Method Article", "title": "Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences", "authors": [ "Charlotte Soneson", "Michael I. Love", "Mark D. Robinson" ], "abstract": "High-throughput sequencing of cDNA (RNA-seq) is used extensively to characterize the transcriptome of cells. Many transcriptomic studies aim at comparing either abundance levels or the transcriptome composition between given conditions, and as a first step, the sequencing reads must be used as the basis for abundance quantification of transcriptomic features of interest, such as genes or transcripts. Several different quantification approaches have been proposed, ranging from simple counting of reads that overlap given genomic regions to more complex estimation of underlying transcript abundances. In this paper, we show that gene-level abundance estimates and statistical inference offer advantages over transcript-level analyses, in terms of performance and interpretability. We also illustrate that while the presence of differential isoform usage can lead to inflated false discovery rates in differential expression analyses on simple count matrices and transcript-level abundance estimates improve the performance in simulated data, the difference is relatively minor in several real data sets. Finally, we provide an R package (tximport) to help users integrate transcript-level abundance estimates from common quantification pipelines into count-based statistical inference engines.", "keywords": [ "RNA-seq", "quantification", "gene expression", "transcriptomics" ], "content": "Introduction\n\nQuantification and comparison of isoform- or gene-level expression based on high throughput sequencing reads from cDNA (RNA-seq) is arguably among the most common tasks in modern computational molecular biology. Currently, one of the most common approaches is to define a set of non-overlapping targets (typically, genes) and use the number of reads overlapping a target as a measure of its abundance, or expression level. Several software packages have been developed for performing such “simple” counting (e.g., featureCounts1 and HTSeq-count2). More recently, the field has seen a surge in methods aimed at quantifying the abundances of individual transcripts (e.g., Cufflinks3, RSEM4, BitSeq5, kallisto6 and Salmon7). These methods provide higher resolution than simple counting, and by circumventing the computationally costly read alignment step, some are considerably faster. However, isoform quantification is more complex than the simple counting, due to the high degree of overlap among transcripts. Currently, there is no consensus regarding the optimal resolution or method for quantification and downstream analysis of transcriptomic output.\n\nAnother point of debate is the unit in which abundance is given. The traditional R/FPKM8,9 (reads/fragments per kilobase per million reads) has been largely superseded by the TPM10 (transcripts per million), since the latter is more consistent across libraries. Regardless, both of these units attempt to “correct for” sequencing depth and feature length and thus do not reflect the influence of these on quantification uncertainty. In order to account for these aspects, most statistical tools for analysis of RNA-seq data operate instead on the count scale. While these tools were designed to be applied to simple read counts, the degree to which their performance is affected by using fractional estimated counts resulting from portioning reads aligning to multiple transcripts is still an open question. The fact that the most common sequencing protocols provide reads that are much shorter than the average transcript length implies that the observed read counts depend on the transcript’s length as well as abundance; thus, simple counts are arguably less accurate measures than TPMs of the true abundance of RNA molecules from given genes. The use of gene counts as input to statistical tools typically assumes that the length of the expressed part of a gene does not change across samples and thus length can therefore be ignored for differential analysis.\n\nIn the analysis of transcriptomic data, as for any other application, it is of utmost importance that the question of interest is precisely defined before a computational approach is selected. Often, the interest lies in comparing the transcriptional output between different conditions, and most RNA-seq studies can be classified as either: 1) differential gene expression (DGE) studies, where the overall transcriptional output of each gene is compared between conditions; 2) differential transcript/exon usage (DTU/DEU) studies, where the composition of a gene’s isoform abundance spectrum is compared between conditions, or 3) differential transcript expression (DTE) studies, where the interest lies in whether individual transcripts show differential expression between conditions. DTE analysis results can be represented on the individual transcript level, or aggregated to the gene level, e.g., by evaluating whether at least one of the isoforms shows evidence of differential abundance.\n\nIn this report, we make and give evidence for three claims: 1) gene-level estimation is considerably more stable than transcript-level; 2) regardless of the level at which abundance estimation is done, inferences at the gene level are appealing in terms of robustness, statistical performance and interpretation; 3) the magnitude of the difference between results obtained by simple counting and transcript-level abundance estimation is generally small in real data sets. However, despite strong overall correlations among results obtained from various quantification pipelines, taking advantage of transcript-level abundance estimates when defining or analyzing gene-level abundances leads to improved differential gene expression results compared to simple counting.\n\nTo facilitate a broad range of analysis choices, depending on the biological question of interest, we provide an R package, tximport, to import transcript lengths and abundance estimates from several popular quantification packages and export (estimated) count matrices and, optionally, average transcript length correction terms (i.e., offsets) that can be used as inputs to common statistical engines, such as DESeq211, edgeR12 and limma13.\n\n\nData\n\nThroughout this manuscript, we utilize two simulated data sets and four experimental data sets (Bottomly14 [Data set 3], GSE6457015 [Data set 4], GSE6924416 [Data set 5], GSE7216517 [Data set 6], see Supplementary File 1 for further details) for illustration. Details on the data generation and full records of the analyses are provided in the data sets and Supplementary File 1. The first simulated data set (sim1; Data set 1) is the synthetic human data set from Soneson et al.18, comprising 20,410 genes and 145,342 transcripts and is available from ArrayExpress (accession E-MTAB-3766). This data set has three biological replicates from each of two simulated conditions, and differential isoform usage was introduced for 1,000 genes by swapping the relative expression levels of the two most dominant isoforms. For each gene in this data set, the total transcriptional output is the same in the two conditions (i.e., no overall DGE); it is worth noting that this is an extreme situation, but provides a useful test set for contrasting DGE, DTU and DTE. The second simulated data set (sim2; Data set 2) is a synthetic data set comprising the 3,858 genes and 15,677 transcripts from the human chromosome 1. It is available from ArrayExpress with accession E-MTAB-4119. Also here, we simulated two conditions with three biological replicates each. For this data set, we simulated both overall DGE, where all transcripts of the affected gene showed the same fold change between the conditions (420 genes), differential transcript usage (DTU), where the total transcriptional output was kept constant but the relative contribution from the transcripts changed (420 genes) and differential transcript expression (DTE), where the expression of 10% of the transcripts of each affected gene was modified (422 genes, 528 transcripts). The three sets of modified genes were disjoint. Again, this synthetic data set represents an extreme situation compared to most real data sets, but provides a useful test case to identify underlying causes of differences between results from various analysis pipelines.\n\n\nGene abundance estimates are more accurate than transcript abundance estimates\n\nTo evaluate the accuracy of abundance estimation with transcript and gene resolution, we used Salmon7 (v0.5.1) to estimate TPM values for each transcript in each of the data sets. Gene-level TPM estimates, representing the overall transcriptional output of each gene, were obtained by summing the corresponding transcript-level TPM estimates. For the two simulated data sets, the true underlying TPM of each feature is known and we can thus evaluate the accuracy of the estimates. Unsurprisingly, gene-level estimates were more accurate than transcript-level estimates (Figure 1A, Supplementary Figures 1,2). We also derived TPM estimates from gene-level counts obtained from featureCounts by dividing each of these with a reasonable measure of the length of the gene (the length of the union of its exons) and the total number of mapped reads, and scaling the estimates to sum to 1 million. The simple count estimates showed a lower correlation with the true TPMs than the Salmon estimates, in line with previous observations19. However, simple counts tended to show a high degree of robustness against incompleteness of the annotation catalog, as evidenced from estimation errors after first removing (at random) 20% of the transcripts (Figure 1A); in contrast, Salmon transcript estimate accuracies deteriorated. From the bootstrap estimates generated by Salmon, we also estimated the coefficient of variation of the abundance estimates. The gene-level estimates showed considerably lower variability in both simulated and experimental data (Figure 1B, Supplementary Figures 3,4). Taken together, these observations suggest that the gene-level estimates are more accurate than transcript-level estimates and therefore potentially allow a more accurate and stable statistical analysis. A further argument in favor of gene-level analysis is the unidentifiability of transcript expression that can result from uneven coverage caused by underlying technical biases (Figure 1C). Intermediate approaches, grouping together “indistinguishable” features are also conceiveable20, but not yet standard practice.\n\nA: Accuracy of gene- and transcript-level TPM estimates from Salmon and scaled FPKM estimates derived from simple counts from featureCounts, in one of the simulated samples (sampleA1). Spearman correlations are indicated in the respective panels. Top row: using the complete annotation. Bottom row: using an incomplete annotation, with 20% of the transcripts randomly removed. Gene-level estimates are more accurate than transcript-level estimates. Gene-level estimates from Salmon are more accurate than those from featureCounts. B: Distribution of the coefficients of variation of gene- and transcript-level TPM estimates from Salmon, calculated across 30 bootstrap samples of one of the simulated samples (sampleA1). Gene-level TPM estimates are less variable than transcript-level estimates. C: An example of unidentifiable transcript-level estimates, as uneven coverage does not cover the critical regions that would determine the amount that each transcript is expressed, while gene-level estimation is still possible.\n\n\nDTE is more powerful and easier to interpret on gene level than for individual transcripts\n\nDTE is concerned with inference of changes in abundance at transcript resolution, and thus invokes a statistical test for each transcript. We argue that this can lead to several complications: the first is conceptual, since the rows (transcripts) in the result table will in many cases not be interpreted independently, but will rather be grouping transcripts from the same gene, and the second one is more technical, since the number of transcripts is considerably larger than the number of genes, which could lead to lower power due to the portioning of the total set of reads across a larger number of features and a potentially higher multiple testing penalty. We tested for DTE on the simulated data by applying edgeR12 to the transcript counts obtained from Salmon (the application of count models to estimated counts is discussed in the next Section), and represented the results as transcript-level p-values or aggregated these to the gene level by using the perGeneQValue function from the DEXSeq21 R package. The transcript-level DTE test assesses the null hypothesis that the individual transcript does not change its expression, whereas the gene-level DTE test assesses the null hypothesis that all transcripts exhibit no change in expression. Framing the DTE question at the gene level results in higher power, without sacrificing false discovery rate control (Figure 2A). We note that this type of gene-level aggregation may favor genes in which one transcript shows strong changes, and that other approaches to increase power against specific alternatives are conceivable, e.g., capitalizing on the rich collection of methods for gene set analysis.\n\nA: DTE detection performance on transcript- and gene-level, using edgeR applied to transcript-level estimated counts from Salmon. The statistical analysis was performed on transcript level and aggregated for each gene using the perGeneQValue function from the DEXSeq R package; aggregated results show higher detection power. The curves trace out the observed FDR and TPR for each significance cutoff value. The three circles mark the performance at adjusted p-value cutoffs of 0.01, 0.05 and 0.1. B: Schematic illustration of different ways in which differential transcript expression (DTE) can arise, in terms of absence or presence of differential gene expression (DGE) and differential transcript usage (DTU).\n\nWhile DTE analysis is more suitable than DGE analysis for detecting genes with changes in absolute or relative isoform expression but no or only minor change in overall output (Supplementary Figure 5), we argue that even gene-level DTE results may suffer from lack of interpretability. DTE can arise in several different ways, from an overall differential expression of the gene or from differential relative usage of its transcripts, or a combination of the two (Figure 2B). We argue that the biological question of interest is in many cases more readily interpretable as a combination of DGE and DTU, rather than DTE. It has been our experience that results reported at the transcript level are still often cast to the gene level (i.e., given a differentially expressed transcript, researchers want to know whether other isoforms of the gene are changing), suggesting that asking two specific gene-level questions (Is the overall abundance changing? Are the isoform abundances changing proportionally?) trumps the interpretability of one broad question at the transcript-level inference (Are there changes in any of the transcript expression levels?). Despite this, there are of course also situations when a transcript-centric approach is superior, for example in targeted experiments where specific isoforms are expected to change due to an administered treatment.\n\n\nIncorporating transcript-level estimates leads to more accurate DGE results\n\nDGE (i.e., testing for changes in the overall transcriptional output of a gene) is typically performed by applying a count-based inference method from statistical packages such as edgeR12 or DESeq211 to gene counts obtained by read counting software such as featureCounts1, HTSeq-count2 or functions from the GenomicAlignments22 R package. A lot has been written about how simple counting approaches are prone to give erroneous results for genes with changes in relative isoform usage, due to the direct dependence of the observed read count on the transcript length23. However, the extent of the problem in real data has not been thoroughly investigated. Here, we show that taking advantage of transcript-resolution estimates (e.g., obtained by Salmon) can lead to improved DGE results. We propose two alternative ways of integrating transcript abundance estimates into the DGE pipeline: to define an “artificial” count matrix, or to calculate offsets that can be used in the statistical modeling of the observed gene counts from, e.g., featureCounts. Both approaches are implemented in the accompanying tximport R package (available from https://github.com/mikelove/tximport).\n\nWe defined three different count matrices for each data set: 1) using featureCounts from the Rsubread1 R package (denoted featureCounts below), 2) summing the estimated transcript counts from Salmon within genes (simplesum), 3) summing the estimated transcript TPMs from Salmon within genes, and multiplying with the total library size in millions (scaledTPM). We note that the scaledTPM values are artificial values, transforming underlying abundance measures to the “count scale” to incorporate the information provided by the sequencing depth. We further used the Salmon transcript lengths and estimated TPMs to define average transcript lengths for each gene and each sample (normalization factors) as described in the Supplementary material, to be used as offsets for edgeR and DESeq2 when analyzing the featureCounts and simplesum count matrices (featureCounts_avetxl and simplesum_avetxl).\n\nOverall, the counts obtained by all methods were highly correlated (Supplementary Figures 6–8), which is not surprising since any differences are likely to affect a relatively small subset of the genes. In general, the simplesum and featureCounts matrices led to similar conclusions in all considered data sets. However, there are differences between the two approaches in terms of how multi-mapping reads and reads partly overlapping intronic regions are handled24. The concordance between simplesum and featureCounts results also suggests that statistical methods based on the Negative Binomial assumption are applicable also to summarized, gene-level estimated counts, which is further supported by the similarity between the p-value histograms as well as the mean-variance relationships observed with the three types of count matrices (Supplementary Figures 9–14).\n\nAccounting for the potentially varying average transcript length across samples when performing DGE, either in the definition of the count matrix (scaledTPM) or by defining offsets, led to considerably improved false discovery rate (FDR) control compared to using the observed featureCounts or aggregated Salmon counts (simplesum) directly (Figure 3A, Table 1). It is important to note that this improvement is entirely attributable to an improved handling of genes with changes in isoform composition between the conditions (Figure 3B, Supplementary Figure 15), that we purposely introduced strong signals in the simulated data set in order to pinpoint these underlying causes, and that the overall effect in a real data set will depend on the extent to which considerable DTU is present. Experiments on various real data sets (Supplementary Figure 16) show only small differences in the collections of significant genes found with the simplesum and simplesum_avetxl approaches, suggesting that the extent of the problem in many real data sets is limited, and that most findings obtained with simple counting are not induced by counting artifacts. Further support for this conclusion is shown in Figure 4 (see also Supplementary Figures 17–19 and Supplementary Table 1), where log-fold change estimates from edgeR, based on the simplesum and scaledTPM matrices, are contrasted. For the genes with induced DTU in the sim2 data set, log-fold changes based on the simplesum matrix are overestimated, as expected. However, this effect is almost absent in all the real data sets, again highlighting the extreme nature of our simulated data and suggesting that the effect of using different count matrices is considerably smaller for many real data sets. Table 1 suggests that the lack of error control for simplesum and featureCounts matrices is more pronounced when there is a large difference in length between the differentially used isoforms. In the group with smallest length difference, where the longer differentially used isoform is less than 34% longer than the shorter one, all approaches controlled the type I error satisfactorily. It is worth noting that among all human transcript pairs in which both transcripts belong to the same gene, the median length ratio is 1.85, and for one third of such pairs the longer isoform is less than 38% longer than the shorter one (see Data set 1).\n\nA: DGE detection performance of edgeR applied to three different count matrices (simplesum, scaledTPM, featureCounts), with or without including an offset representing the average transcript length (for simplesum and featureCounts). Including the offset or using the scaledTPM count matrix leads to improved FDR control compared to using simplesum or featureCounts matrices without offset. The curves trace out the observed FDR and TPR for each significance cutoff value. The three circles mark the performance at adjusted p-value cutoffs of 0.01, 0.05 and 0.1. B: stratification of the results in A by the presence of differential isoform usage. The improvement in FDR control seen in A results from an improved treatment of genes with differential isoform usage, while all methods perform similarly for genes without differential isoform usage.\n\nFor the simulated data set (sim2), where signals have been exaggerated to pinpoint underlying causes of various observations, genes with induced DTU (whose true overall log-fold change is 0) show a clear overestimation of log-fold changes when using simplesum counts. However, none of the real data sets contain a similar population of genes, suggesting that for many real data sets, simple gene counting leads to overall similar conclusions as accounting for underlying changes in transcript usage.\n\nThe results are stratified by “effect size” (the difference in relative abundance between the two differentially used isoforms) and the length ratio between the longer and the shorter of the differentially used isoforms. FPRs below the nominal p-value threshold (0.05) are marked in bold. For more details, see Data set 1.\n\n\nDiscussion\n\nIn this article, we have contrasted transcript- and gene-resolution abundance estimation and statistical inference, and illustrated that gene-level results are more accurate, powerful and interpretable than transcript-level results. Not surprisingly, however, accurate transcript-level estimation and inference plays an important role in deriving appropriate gene-level results, and it is therefore imperative to continue improving abundance estimation and inference methods applicable to individual transcripts, since misestimation can propagate to the gene level. We have shown that when testing for changes in overall gene expression (DGE), traditional gene counting approaches may lead to an inflated false discovery rate compared to methods aggregating transcript-level TPM values or incorporating correction factors derived from these, for genes where the relative isoform usage differs between the compared conditions. These correction factors can be calculated from the output of transcript abundance programs, using e.g., the provided R package (tximport). It is important to note that the average transcript length offsets must account for the differences in transcript usage between the samples and thus using (sample-independent) exon-union gene lengths will not improve performance.\n\nAll evaluated counting approaches gave comparable results for genes where DTU was not present. Thus, the extent of the FDR inflation in experimental data depends on the extent of DTU between the compared conditions; notably, our simulation introduced rather extreme levels of DTU, hence the inflated FDR, and the difference between the approaches was considerably smaller in real data sets. Recent studies have also shown that many genes express mainly one, dominant isoform25 and for such genes, we expect that simple gene counting will work well.\n\nOur results highlight the importance of correctly specifying the question of interest before selecting a statistical approach. Summarization of abundance estimates at the gene level before performing the statistical testing should be the method of choice if the interest is in finding changes in the overall transcriptional output of a gene. However, it is suboptimal if the goal is to identify genes for which at least one of the transcripts show differences in transcriptional output, since it may miss genes where two transcripts change in opposite directions, or where a lowly expressed transcript changes. For gene-level detection of DTE (that is, whether any transcript showed a change in expression between the conditions), statistical testing applied to aggregated gene counts led to reduced power and slightly inflated FDR compared to performing the statistical test on the transcript level and aggregating results within genes (Supplementary Figure 5). Statistical inference on aggregated transcript TPMs (scaledTPM) showed low power for detecting changes that did not affect the overall transcriptional output of the gene, as expected. An alternative to DTE analysis, for potential improved interpretability, is to perform a combination of DGE and DTU analyses, both resulting in gene-level inferences. Table 2 summarizes our results and give suggested workflows for the different types of analyses we have considered.\n\nOf course, there may be situations where a direct transcript-level analysis is appropriate. For example, in a cancer setting where a specific deleterious splice variant is of interest (e.g., AR-V7 in prostate cancer26), inferences directly at the transcript level may be preferred. However, while this may be preferred for individual known transcripts, transcriptome-wide differential expression analyses may not be warranted, given the associated multiple testing cost.\n\nFinally, we note that estimation at the gene level can reduce the problem of technical biases on expression levels and unidentifiable estimation. Current methods for transcript-level quantification (e.g., Cufflinks, RSEM, Salmon, kallisto) do not correct for amplification bias on fragments, which can lead to many estimation errors, such as expression being attributed to the wrong isoform27. Non-uniform coverage from amplification bias or from position bias (3’ coverage bias from poly-(A) selection) can result in unidentifiable transcript-level estimation. Such errors and estimation problems are minimized when summarizing expression to the gene level.\n\n\nData availability\n\nF1000Research: Data set 1. 10.5256/f1000research.7563.d109328\n\nF1000Research: Data set 2. 10.5256/f1000research.7563.d109329\n\nF1000Research: Data set 3. 10.5256/f1000research.7563.d109330\n\nF1000Research: Data set 4. 10.5256/f1000research.7563.d109331\n\nF1000Research: Data set 5. 10.5256/f1000research.7563.d109332\n\nF1000Research: Data set 6. 10.5256/f1000research.7563.d109333\n\n\nSoftware availability\n\nhttps://github.com/mikelove/tximport\n\nhttps://github.com/F1000Research/tximport\n\nhttp://dx.doi.org/10.5281/Zenodo.35123\n\ntximport is released under a GNU Public License (GPL).", "appendix": "Author contributions\n\n\n\nCS, MIL and MDR conceived the study and developed methodology. CS and MDR designed and carried out the computational experiments and drafted the manuscript. MIL implemented the tximport R package and wrote parts of the manuscript. All authors read and approved the final manuscript and have agreed to the content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nMDR and CS acknowledge support from the “RNA & Disease” National Center of Competence in Research, an SNSF project grant (143883) and from the European Commission through the 7th Framework Collaborative Project RADIANT (Grant Agreement Number: 305626). MIL was supported by NIH grant 5T32CA009337-35.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nThe authors would like to thank Magnus Rattray, Alexander Kanitz, Hubert Rehrauer and Xiaobei Zhou for helpful comments on earlier versions of this manuscript.\n\n\nSupplementary material\n\nSupplementary File 1.\n\nSupplementary File 1 (pdf) contains more detailed information about the data sets, supplementary methods and supplementary figures referred to in the text.\n\nClick here to access the data.\n\n\nReferences\n\nLiao Y, Smyth GK, Shi W: featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2014; 30(7): 923–30. PubMed Abstract \| Publisher Full Text\n\nAnders S, Pyl PT, Huber W: HTSeq - a Python framework to work with high-throughput sequencing data. Bioinformatics. 2015; 31(2): 166–169. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTrapnell C, Roberts A, Goff L, et al.: Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc. 2012; 7(3): 562–78. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi B, Dewey CN: RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics. 2011; 12: 323. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGlaus P, Honkela A, Rattray M: Identifying differentially expressed transcripts from RNA-seq data with biological variation. Bioinformatics. 2012; 28(13): 1721–1728. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBray N, Pimentel H, Melsted P, et al.: Near-optimal RNA-Seq quantification. arXiv:1505.02710. 2015. Reference Source\n\nPatro R, Duggal G, Kingsford C: Accurate, fast, and model-aware transcript expression quantification with Salmon. bioRxiv. 2015. Publisher Full Text\n\nMortazavi A, Williams BA, McCue K, et al.: Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods. 2008; 5(7): 621–628. PubMed Abstract \| Publisher Full Text\n\nTrapnell C, Williams BA, Pertea G, et al.: Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol. 2010; 28(5): 511–515. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWagner GP, Kin K, Lynch VJ: Measurement of mRNA abundance using RNA-seq data: RPKM measure is inconsistent among samples. Theory Biosci. 2012; 131(4): 281–285. PubMed Abstract \| Publisher 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\nRitchie ME, Phipson B, Wu D, et al.: limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015; 43(7): e47. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBottomly D, Walter NA, Hunter JE, et al.: Evaluating gene expression in C57BL/6J and DBA/2J mouse striatum using RNA-Seq and microarrays. PLoS One. 2011; 6(3): e17820. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYang S, Marín-Juez R, Meijer AH, et al.: Common and specific downstream signaling targets controlled by Tlr2 and Tlr5 innate immune signaling in zebrafish. BMC Genomics. 2015; 16(1): 547. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCurrais A, Goldberg J, Farrokhi C, et al.: A comprehensive multiomics approach toward understanding the relationship between aging and dementia. Aging (Albany. NY). 2015; 7(11): 937–955. PubMed Abstract\n\nChang AJ, Ortega FE, Riegler J, et al.: Oxygen regulation of breathing through an olfactory receptor activated by lactate. Nature. 2015; 527(7577): 240–244. PubMed Abstract \| Publisher Full Text\n\nSoneson C, Matthes KL, Nowicka M, et al.: Differential transcript usage from RNA-seq data: isoform pre-filtering improves performance of count-based methods. bioRxiv. 2015. Publisher Full Text\n\nKanitz A, Gypas F, Gruber AJ, et al.: Comparative assessment of methods for the computational inference of transcript isoform abundance from RNA-seq data. Genome Biol. 2015; 16(1): 150. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRobert C, Watson M: Errors in RNA-Seq quantification affect genes of relevance to human disease. Genome Biol. 2015; 16: 177. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAnders S, Reyes A, Huber W: Detecting differential usage of exons from RNA-seq data. Genome Res. 2012; 22(10): 2008–17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLawrence M, Huber W, Pagès H, et al.: Software for computing and annotating genomic ranges. PLoS Comput Biol. 2013; 9(8): e1003118. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTrapnell C, Hendrickson DG, Sauvageau M, et al.: Differential analysis of gene regulation at transcript resolution with RNA-seq. Nat Biotechnol. 2013; 31(1): 46–53. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhao S, Xi L, Zhang B: Union Exon Based Approach for RNA-Seq Gene Quantification: To Be or Not to Be? PLoS One. 2015; 10(11): e0141910. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGonzàlez-Porta M, Frankish A, Rung J, et al.: Transcriptome analysis of human tissues and cell lines reveals one dominant transcript per gene. Genome Biol. 2013; 14(7): R70. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAntonarakis ES, Lu C, Wang H, et al.: AR-V7 and resistance to enzalutamide and abiraterone in prostate cancer. N Engl J Med. 2014; 371(11): 1028–38. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLove MI, Hogenesch JB, Irizarry RA: Modeling of RNA-seq fragment sequence bias reduces systematic errors in transcript abundance estimation. bioRxiv. 2015. Publisher Full Text\n\nSoneson C, Love MI, Robinson MD: Data set 1 in: Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences. F1000Research. 2015. Data Source\n\nSoneson C, Love MI, Robinson MD: Data set 2 in: Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences. F1000Research. 2015. Data Source\n\nSoneson C, Love MI, Robinson MD: Data set 3 in: Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences. F1000Research. 2015. Data Source\n\nSoneson C, Love MI, Robinson MD: Data set 4 in: Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences. F1000Research. 2015. Data Source\n\nSoneson C, Love MI, Robinson MD: Data set 5 in: Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences. F1000Research. 2015. Data Source\n\nSoneson C, Love MI, Robinson MD: Data set 6 in: Differential analyses for RNA-seq: transcript-level estimates improve gene-level inferences. F1000Research. 2015. Data Source" }	[ { "id": "11761", "date": "04 Jan 2016", "name": "Stephen N. Floor", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSoneson, Love and Robinson tackle a crucial question for analysis of RNA deep sequencing data in this manuscript: what is the role of transcript diversity in the accuracy and statistical power associated with measurements of gene expression? The authors make and convincingly show three claims: gene-level estimation and inferences are more robust than those at the transcript-level, and incorporating transcript-level quantification into gene-level abundance leads to improved differential expression testing. The claims are convincingly proven, the manuscript is well written, and the subject matter is of considerable interest. Furthermore, the described R package tximport should be of broad interest to the RNA deep sequencing community. Overall comments: It may be useful to indicate explicitly in the text that the methods are contained within the (excellently written and formatted) supplementary material, as this was not apparent. It might be clearest to create a specific methods section that just references supplementary file 1. The clarity of scatter plots with more than ~hundreds of points (e.g. Figure 1A) could be improved by using partially transparent points to visualize density. Introduction: Paragraph 1: Cufflinks, RSEM and Bitseq are grouped with kallisto and Salmon and it is then stated that some of these methods bypass read alignment. It would be clearer if this were reworded to avoid the ambiguity as to which methods avoid read alignment. Paragraph 4: The third claim could be presented more clearly. While it is interesting that simple counting performs similarly to transcript-level quantification procedures, it seems more interesting to this reviewer that incorporating transcript-level information improves the accuracy of differential expression testing at the gene level. Perhaps these two concepts can be combined into one more concise point? Results: The assertion that “simple counts tended to show a high degree of robustness against incompleteness of the annotation catalog, as evidenced from estimation errors after first removing (at random) 20% of the transcripts” seems misleading since Salmon-derived gene-level abundances actually show a higher Spearman correlation than count-derived gene-level abundances when subjected to removing a random 20% of transcripts. Figure 1a bottom left shows that transcript-level abundances are strongly affected by removal of 20% of transcripts, but that gene-level abundances are not strongly changed whether estimated using counts or Salmon. This statement should be reworded. Two concerns are raised about DTE. It is certainly true that reads are spread across more features when performing DTE as opposed to DGE.However, it is not apparent why analysis of DTE involves grouping of transcripts together for interpretation. DTE implies analysis at the transcript level and therefore no grouping, while DGE could involve some level of grouping of transcripts or quantification at the gene level from the start. The clarity of this could be improved. It is a very interesting idea to separately frame questions regarding DGE and DTU, which should be adopted widely, as the two are separable questions. The authors state one possible workflow towards DGE analysis in the section “Incorporating transcript-level estimates leads to more accurate DGE results.” Alternative pipelines (e.g. cuffdiff) could be presented in brief. The observation that simplesum and featureCounts results are highly correlated and therefore that statistical methods based on the Negative Binomial distribution can be used on estimated counts seems of greater importance than is emphasized in the text. This should be elaborated upon in the discussion, since this means that estimated counts from kallisto, express, salmon, etc can be used directly by statistical packages assuming a NB distribution (edgeR, DESeq2, etc). This point is frequently debated in discussions of how to rigorously analyze sequencing data. The conclusion here that NB applies to estimated counts is thus quite important. Please explain the meaning of the name for each curve in the legend for Figure 3 (i.e. specify that “avetxl” means using the offset corresponding to average transcript length. Discussion: The assertion that “gene-level results are more accurate, powerful and interpretable than transcript-level results” seems an oversimplification given the result that incorporating transcript-level quantification leads to improved DGE detection performance (e.g. Fig 3). Please cite at minimum Roberts et al., (2011) regarding sequence bias correction as this has been implemented in cufflinks, express and kallisto. Other relevant papers should also be included here, as attempts have been made to address both positional and sequence-specific bias in RNA sequencing data. Supplement: The usability of the supplemental info could be improved by substituting rasterized for vectorized plots for those with ~hundreds of points. Please explain the meaning of the name for each curve in the legend for Supplemental Figure 5.", "responses": [ { "c_id": "1821", "date": "29 Feb 2016", "name": "Mark Robinson", "role": "Author Response", "response": "Thank you for taking the time to read and review our paper. In the revised version, we have improved the clarity and usability of the figures and the supplement by using partially transparent points and extending the figure captions. We have also modified the main text to refer more clearly to the method information provided in the supplementary material, and improved the clarity of the text in several places, according to your suggestions. For additional comments regarding references to bias correction studies, please see responses to Rob Patro." } ] }, { "id": "11745", "date": "12 Jan 2016", "name": "Rob Patro", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 address a few questions of considerable (and perennial) interest in the analysis of RNA-seq data. Specifically, they provide evidence that, using available methods (e.g. DESeq2 / edgeR), assessing differential expression at the gene-level (DGE) is more robust than at the transcript level (DTE). Further, they convincingly argue that estimating abundance at the level of transcripts, and then aggregating these abundances to the gene level leads to improved estimation of differential gene expression. They demonstrate that one of the major factors in this improved estimation is the availability of a sample-specific feature length for each gene (derived from the abundance-weighted length of the expressed transcripts of this gene), which is not possible to obtain with any fixed gene model used by count-based methods. Finally, the authors argue that much of the analysis of interest at the transcript level does not actually require differential transcript expression testing, but rather can be inferred from a combination of DGE and differential transcript usage (DTU); this is an interesting proposition that merits further discussion and analysis. Overall, this is a well-written paper, with extensive and compelling supplementary and supporting data, that addresses a ubiquitous analysis task involving RNA-seq. It should be of broad interest to the community and makes a valuable contribution. The accompanying software, tximport, is user-friendly and makes it easy to apply the type of analysis recommended herein; it too should be widely useful.Major comments:It would be very useful to provide the equations used for calculating each of the abundance measured considered directly. Section 4 of the supplementary information is useful to this end, but the reader still has to search a bit to see exactly how each metric is computed (though the fantastic R-Markdown included with the figures means that these computations can be found explicitly).Similarly, it would be useful to the reader to provide a description, in prose, of how specific experiments were performed (again, the reproducible nature of most of these experiments makes tracking down this information possible, but sometimes time-consuming). For example, how, precisely, was removal of transcripts handled at the level of the genome annotation? If a transcript consists only of constitutive exons, were all of those exons retained in the genome annotation used for STAR + featureCounts, while the transcript was removed in the Salmon index? The result that transcript-level abundance estimation is more sensitive to the removal of transcripts than gene-level abundance estimation — this seems intuitive. However, I agree with Dr. Floor’s suggestion that:\"The assertion that “simple counts tended to show a high degree of robustness against incompleteness of the annotation catalog, as evidenced from estimation errors after first removing (at random) 20% of the transcripts” seems misleading since Salmon-derived gene-level abundances actually show a higher Spearman correlation than count-derived gene-level abundances when subjected to removing a random 20% of transcripts.”I would suggest rewording this sentence, as the main result seems to be that gene-level analysis is more robust to an incomplete annotation than transcript-level analysis. Transcript-level abundance estimation followed by gene-level analysis seems to perform just as well (actually, better) than gene-level counting in this scenario.The experiments in the section “Incorporating transcript-level estimates leads to more accurate DGE results” suggests the (reasonable) interpretation that the main benefit of incorporating transcript-level abundance estimates when assessing DGE is a more accurate measure of the “feature” length of the gene. The authors state “ It is important to note that this improvement is entirely attributable to an improved handling of genes with changes in isoform composition between the conditions.” This is supported by the fact that using the abundance-weighted average transcript length (i.e. offsets) with counting based approaches improves the results substantially. However, one other place where transcript-level abundance estimates are useful in the context of DGE is when assessing the expression of paralogous genes. While most multi-mapping reads that derive from different isoforms of the same gene will be uniquely mappable at the level of the genome, and hence will be included in the counts for that gene, reads that map ambiguously among paralogs may not be. In such cases, count-based methods do not have a principled way of apportioning a read between the paralogs involved, and discarding multi-mapping reads may negatively affect estimation of the abundance of the paralogs, even at the gene level. While this case is likely much less common than mis-estimation of DGE as a result of DTU, it is certainly of biological interest. I would suggest adding an analysis, restricted to sets of paralogous genes, comparing how the different approaches perform in this case. This may help to highlight the importance of not only deriving appropriately weighted and sample-dependent lengths for genes, but also on resolving multi-mapping ambiguity that occurs between genomically distinct loci.The argument that most transcript-level analyses of interest may be addressed by looking at DGE in conjunction with DTU is an interesting one. It is certainly that case that not all tasks for which DTE is used actually require assessing differential expression at the transcript level. One issue with the DGE + DTU-based analysis which warrants further discussion in the manuscript is that I believe that this approach, too, would require correcting for multiple hypothesis testing. Specifically, one is testing both the DGE and the DTU hypotheses for each gene (or for a relevant subset of interest). The correction here is likely to be less harsh than in the case of assessing DTE, but is still worth discussing.Minor comments:As per Dr. Floor’s statement, Salmon (and Sailfish) also incorporate sequence-specific bias correction. Further, RSEM and Salmon (and a few other transcript-level abundance estimation tools) also incorporate the modeling of non-uniform fragment start position distributions. Of course, modeling a non-uniform start position distribution cannot overcome a complete lack of sampling in critical regions that might make determining transcript-level fragment assignment impossible, but it may help in properly apportioning an ambiguously-mapped fragment between transcripts depending on its relative position in each.One potential added source of variability here is that all Salmon estimates presented in the manuscript make use of Salmon’s quasi-mapping of reads, while the STAR + featureCount pipeline makes use of “traditional” alignments. This is the primary intended usage mode of Salmon, and absolutely does represent a “typical” pipeline for methods that avoid alignment (Salmon, Sailfish, kallisto). However, it would probably be best to mention this as a potential (though likely negligible) additional source of variability.In the discussion, the authors argue that “... it is therefore imperative to continue improving abundance estimation and inference methods applicable to individual transcripts, since misestimation can propagate to the gene level.” This is, of course, an important and valid suggestion. Another direction, on which it would be useful to get the authors’ thoughts and suggestions, is the development of differential expression tools (at either the transcript or gene level) that can make use of the variance estimates that some tools (like Salmon) can provide. To what extent might incorporating this information help control false positive rates and improve DTE or even DGE estimates?", "responses": [ { "c_id": "1820", "date": "29 Feb 2016", "name": "Mark Robinson", "role": "Author Response", "response": "Thank you for taking the time to read and review our paper. As per your suggestions, we have expanded the supplementary pdf document with equations defining each of the abundance measures that we used as well as a detailed description of the generation of the incomplete annotation files. We have also reworded the paragraph discussing the influence of an incomplete annotation catalog, and added further discussion points regarding multiple testing correction for DGE+DTU as well as regarding new methods incorporating variance estimates into the differential analysis. However, a deeper discussion of the latter point falls outside the scope of this article. We found your suggestion to restrict the abundance accuracy comparison to paralogous genes interesting, and the supplement has been expanded with several examples comparing the accuracy of gene-level abundance estimates from Salmon and from STAR+featureCounts, restricted to sets of paralogous genes. While we see a clear advantage of Salmon for many of the paralogous gene groups, the overall difference is only slightly larger for sets of paralogous genes compared to random sets of genes (as can be seen in the revised version of Dataset 1). In addition, we have added the following text to the relevant section in the results: “...suggesting that the extent of the problem in many real data sets is limited... though some loss of sensitivity for certain genes may be encountered from discarding multi-mapping fragments, which may be recovered through the use of transcript abundance estimators such as Salmon.” While we acknowledge that many of the popular transcript abundance estimation methods incorporate some type of bias estimation/correction (albeit not at the fragment level), and that it is definitely an important (and difficult) research area, a thorough discussion on the relative merits of different bias correction approaches is outside the scope of this paper. We have added citations to relevant literature on coverage bias, have simplified and clarified the last paragraph of the discussion, and have reworded the sentence about unidentifiable estimation due to coverage biases: “While some extent of coverage variability might be alleviated by corrections for sequence- or position-specific biases, there remain cases where transcript expression cannot be inferred from data ( Figure 1C)”" } ] } ]	1	https://f1000research.com/articles/4-1521
https://f1000research.com/articles/5-238/v1	29 Feb 16	{ "type": "Review", "title": "Tumour Cell Heterogeneity", "authors": [ "Laura Gay", "Ann-Marie Baker", "Trevor A. Graham" ], "abstract": "The population of cells that make up a cancer are manifestly heterogeneous at the genetic, epigenetic, and phenotypic levels. In this mini-review, we summarise the extent of intra-tumour heterogeneity (ITH) across human malignancies, review the mechanisms that are responsible for generating and maintaining ITH, and discuss the ramifications and opportunities that ITH presents for cancer prognostication and treatment.", "keywords": [ "Cancer evolution", "intra-tumour heterogeneity", "evolutionary biomarkers", "personalised medicine" ], "content": "The origins of intra-tumour heterogeneity\n\nIntra-tumour heterogeneity (ITH) has been documented for many decades, initially from a morphological perspective1,2. Cancers of all types are now recognised to consist of highly diverse populations of cells3, where ITH is detectable at the genetic, epigenetic, and phenotypic levels (see Table 1 for a pan-cancer summary). Recent advances in next-generation sequencing and microarray technology have enabled researchers to begin to appreciate the full extent and complexity of ITH. As a major cause of targeted therapy failure and disease resistance4, ITH is a subject of much biological and clinical relevance.\n\nWhen viewed through the lens of evolutionary biology, the sometimes extreme levels of diversity present in cancers5 should come as no surprise6. Carcinogenesis is an evolutionary process whereby somatic cells acquire random (epi)mutations that alter their phenotype, and the fittest new clones clonally expand because of the action of Darwinian natural selection7; repeated rounds of mutation and natural selection can lead to the development of a malignant cancer clone that is capable of migration and growth in remote sites. Diversity in the evolving cancer ecosystem is inevitable because it fuels the evolutionary fire; there can be no “survival of the fittest” if all the cells in the tumour have the same fitness.\n\nThere are many mechanisms that contribute to ITH, and these can be broadly classified as “cell autonomous” or “non-cell autonomous”. An example of a cell-autonomous mechanism is the persistence of small numbers of errors that occur during DNA replication43,44. When multiplied by the billions of cell divisions required to produce even the smallest clinically detectable tumour (with a volume of approximately 1 cm3), this low level of mutation can potentially generate tremendous within-tumour genetic diversity. Moreover, the rate at which diversity is generated in a tumour is typically accelerated by genetic instability, likely a consequence of replication stress45, and the “mutator phenotype”46 that is a feature of most solid tumours. Furthermore, there can be rare but catastrophic DNA replication errors occurring during a single mitosis that can lead to the production of daughter cells with grossly altered genomes32,47,48.\n\nA result of recent advances in next-generation sequencing is that single-cell whole genome sequencing is now possible (and indeed transcriptome sequencing too)49. Therefore, it is conceivable that we will soon be able to envisage mapping the genetic diversity of an entire tumour at cellular resolution. However, an important question is how much of this information about genetic ITH will prove to be clinically relevant? Intriguingly, in some cancers (lung10,11 and colon13,14), the key driver mutations are proven to be clonal (e.g. present in all tumour cells), although spatially localised drivers have been found in other cancer types (e.g. kidney9). The obvious question then becomes how many, if indeed any, of the heterogeneous mutations are important for tumour growth? Clearly, there is a need to discriminate between ITH that is attributable to “mutational noise” (e.g. the background mutation rate) and that which is in some sense “functional” for tumour development. Mathematical modelling of the amount of ITH that should be expected in a growing tumour can be helpful here, as an increase or decrease of ITH compared with expectation reveals “important” evolution in the tumour (our effort to implement such a model50 is discussed briefly below). An alternative empirical approach would require the concurrent measurement of genotype and phenotype so that genetic ITH can be related to the presence of (minority) cell populations with biologically distinct function. Interestingly, measurement of the behaviour of different clones within a colon cancer, including their sensitivity to cytotoxic drugs, revealed marked differences in behaviour between tumour regions, without concomitant differences in genotype15. This study in particular highlights how genetic ITH can be a poor proxy for functional heterogeneity, and the latter is clearly of greater clinical relevance, especially ITH of drug response.\n\nA further source of ITH is the persistence of a cancer stem cell (CSC) hierarchy, a factor that may be described as either cell-autonomous (i.e. “stemness” is governed by a cell’s genetic or epigenetic makeup) and non-cell autonomous (i.e. “stemness” is governed by external factors within an environmental “niche”). Although a strict hierarchy of differentiation is described as a feature of haematopoietic cancers51, a form of CSC architecture may significantly contribute to ITH in solid tumours. There is evidence of a CSC hierarchy in mouse models of brain cancer52,53, where a small subpopulation of cells is responsible for sustaining tumour growth. The definition of a CSC continues to be debated54, but if CSCs are defined simply as the population of cells with long-term self-renewal capability, then following debulking chemotherapy tumour regrowth is determined by the prevalence of the CSC phenotype amongst the surviving cells18. The question of the plasticity of the stem cell phenotype is an interesting topic of discussion. It is noteworthy that observations of cell populations growing in vitro55 and genetically engineered mouse models of intestinal tumourigenesis56 suggest that bidirectional phenotype switching can occur (even in the absence of clonal selection), questioning whether the “stemness” of a tumour cell is strictly intrinsically defined. Plasticity of a CSC phenotype can be viewed as a form of epigenetic heterogeneity within the tumour.\n\nHeterogeneity in the microenvironment of a cancer can be described as a non-cell autonomous driver of cancer cell diversity62; in a highly diverse microenvironment, different cellular phenotypes may be selected for or against in different regions of the tumour. For example, any sizeable tumour will inevitably contain areas of hypoxia and normoxia. In a hypoxic region, an anaerobically metabolising cell is expected to have a fitness advantage over an aerobically metabolising cell and so should repopulate the hypoxic region, but the opposite should be true in a normoxic region63. Such different phenotypes may be genetically determined (such as the case of VHL/HIF1 mutants in kidney cancer)64, or they may be a consequence of plasticity in cellular behaviours15, and furthermore there may be feedback between the tumour cells and their microenvironment that drives specialisation of tumour cells and the concomitant strengthening of microenvironmental gradients.\n\nA further example of a non-cell autonomous driver of ITH is the interactions between subclones of the tumour. In experimental systems, synergistic or predatory interactions between phenotypically distinct clones drive tumour growth15,58,59,65–67. Intermixing of clonally distinct populations is a feature of many tumours and has been particularly well documented in gliomas16. If interactions between cells within a tumour are critical for tumour maintenance, then reducing the tumour population size below some critical threshold—called an Allee threshold—may be an effective cancer treatment61. The logic behind this idea is that small populations are not able to produce sufficient density of cooperative factors that are necessary for tumour maintenance (such as diffusible growth factors) and so small populations are unviable. The interplay between clones that are producers (e.g. angiogenic cells) versus consumers (e.g. aerobically respiring non-angiogenic cells) should, theoretically, also influence phenotypic diversity within a cancer. Clearly, tumour cells can also benefit from factors produced by the stromal cells in their microenvironment68. Clonal interference refers to the situation where two or more clones of similar fitness each impede the growth of the other by competing equally well for limited resources, and theoretical models of cancer growth predict that clonal interference should slow cancer evolution and lead to the longer-term maintenance of multiple distinct clonal populations69. Interactions between tumour cells and the immune system also shape ITH: for example, the immune system predates the tumour cells, and tumour cells that have evolved to avoid immune detection will persist and perhaps clonally expand in the tumour70.\n\nRecognising that there is a dynamic interplay between tumour cells and their microenvironment, and between tumour cells themselves, means that cancers are best viewed as complex evolving ecosystems60. In the cancer ecosystem, the relative fitness conferred by a new mutation is defined by microenvironmental context6,57, where the context refers to both the neighbourhood of tumour and stromal cells and more broadly diffusible factors. Importantly, the ecological viewpoint provides a single framework to understand the seemingly distinct contributions of cell-autonomous and non-cell autonomous factors to ITH. This is because the ecological view forces us to recognise that all the evolution within a tumour is only ever driven by selection in the current microenvironment context: cell-autonomous drivers provide an advantage to the cell in their current context, and non-cell autonomous factors can drive evolution by changing that context.\n\nMulti-faceted networks are challenging to understand, and consequently computational or mathematical models are increasingly recognised as essential tools to integrate and interpret the complex, multi-scale data derived from the interplay between tumour subclones and their interactions with the microenvironment (ref. 71 provides an overview). Part of the value of mathematical models is the ability to elucidate the underlying (perhaps simple) causes of intricate patterns in complex systems. For example, our own work has used a simple mathematical model of mutation during the first few rounds of cell doubling at the start of tumour growth to explain the complex pattern of genetic ITH observed in cancer50. We note that this kind of approach can help to delineate “important” ITH from the inconsequential “mutational noise” mentioned above. More generally, such models can be used to generate new hypotheses concerning the generation and maintenance of ITH, and the effects of ITH on tumour evolution, and to predict response to therapy and recurrence.\n\nIn summary, the fact that tumours are an evolving ecosystem means that ITH is inevitable, and consequently ITH is observed in all tumour types and premalignant diseases where it has been looked for. We devote the remainder of this review to understanding the consequences of ITH for cancer prognostication and treatment.\n\n\nIntra-tumour heterogeneity and prognostication\n\nTraditionally, cancer prognosis has been determined by the presence or absence of a particular feature within a tumour. Histopathology remains the mainstay of this approach: tumours are scored for stage (how far the cancer has invaded) and grade (a measure of how abnormal the cancer cells appear and how disrupted the tissue architecture of the tumour is compared with normal tissue). Molecular markers have entered clinical practice too, most prominently assessment of oestrogen receptor (ER), progesterone receptor (PR), and HER2 status in breast cancer73,74 (to determine prognosis and treatment choice). Relatedly, genetic analysis is employed to predict the likely efficacy of targeted therapies by testing whether the tumour contains pre-existing resistant clones; for example, colorectal cancers are screened for KRAS mutations as the presence of a KRAS mutant clone means that the anti-epidermal growth factor receptor (anti-EGFR) antibody cetuximab will be an ineffective treatment75.\n\nClearly, ITH presents a major obstacle for such “feature-based” prognostic markers, simply because if the feature being assayed for is not present in the particular biopsy analysed but is present elsewhere in the tumour then prognosis will be incorrectly assigned (Figure 1A). A striking demonstration of this was provided by Gerlinger and colleagues when they compared gene expression-based prognostic signatures derived from spatially distinct regions on a single renal cancer and reported that the different regions of the same tumour can harbour either good- or bad-prognosis signatures; therefore, a single biopsy would not sufficiently represent the tumour composition8. Epistatic interactions (perhaps driven by unmeasured genes) could also potentially alter the prognostic value of individual molecular feature (Figure 1C). There is also an issue of what feature (or indeed set of features) to include in a prognostic assay. One of the striking findings of the recent large-scale extensive molecular characterisations of tumours, such as The Cancer Genome Atlas project (http://cancergenome.nih.gov), is that every tumour appears molecularly unique or, in other words, that there appear to be many different ways to produce a particular cancer. This rampant inter-tumour heterogeneity may preclude a “one size fits all” approach to feature-based prognostic markers since a particular prognostic feature is unlikely to work for all cancers of a particular type (Figure 1B). Relatedly, the differences between cancers of distinct types may mean that a particular feature is unlikely to have broad prognostic value (Figure 1D), although there is evidence suggesting that integral biochemical cellular features such as the ability to form proper DNA segregation machinery77,78 or to properly regulate adhesion79 may have potential in this respect.\n\n(A) Intra-tumour heterogeneity means a targeted biopsy may miss a lesion with a poor prognostic signature (“red” phenotype) within the tumour (majority of tumour has a “blue” phenotype that is associated with a good prognosis). (B) Differences between tumours (inter-tumour heterogeneity) mean that a single prognostic biomarker may be unsuitable for use in some tumours that have evolved along a different carcinogenic pathway; this yellow tumour does contain either the previously identified good (blue) or bad (red) phenotypes. (C) Epistasis between genes (or other intra- or inter-cellular interactions) can alter the prognostic value of any individual feature; here, the presence of the “green” mutation may alter the bad prognosis of the “red” mutation assayed in (A). (D) Inter-cancer heterogeneity means that a feature-based prognostic marker developed for one cancer type is unlikely to work in another cancer type.\n\nThe idea that some molecular changes may be integral to tumour biology, and so not subject to ITH, is supported by an intriguing study of renal cancers that provides a counterpoint to the findings of Gerlinger and colleagues. Rini and colleagues used a large cohort of renal cancers to derive a 16-gene signature that predicted recurrence76. Importantly, the authors also performed multi-region sampling on a small number of samples to demonstrate little or no ITH in their gene signature, which, counter to the findings of Gerlinger and colleagues, would suggest that sampling one biopsy can in fact be enough to infer prognosis. In broad terms, the prognostic importance of ITH is likely to be dependent on many factors such as tumour type, tumour stage, and treatment regime, and critically the nature of the biomarker itself.\n\nHowever, it is not all doom and gloom. Approaching ITH from an evolutionary perspective suggests a new approach to prognostication that exploits ITH rather being hindered by it. The idea is that the evolvability of a population is determined (somewhat) by the degree of diversity present in that population (Figure 2). To understand this idea, suppose there are two populations of cells: one where all the cells are identical to one another (low diversity) and one where all the cells are phenotypically distinct (high diversity). If the cells in the first scenario experience a new selective pressure (e.g. a cytotoxic drug), then either all the cells are perfectly adapted and nothing happens, or the population is eradicated. When the same selective pressure is applied in the second scenario, the sensitive cells in the phenotypically diverse population will be killed off and any (perhaps minor) subclone that was resistant to the pressure will survive and grow to dominate the tumour in the absence of competing clones. Therefore the idea is that more diverse tumours are more likely to be more evolvable and hence more likely to generate a metastatic clone or contain a clone that is resistant to therapy (or both): hence, the theory is that tumours with higher levels of ITH should have a worse prognosis.\n\nA homogeneous tumour (A) will be eradicated in response to a selective pressure such as chemotherapy, whereas a heterogeneous tumour (B) is more likely to contain a pre-existing resistant clone that survives the selective pressure and seeds the repopulation of the tumour.\n\nEmpirical studies support the idea of quantification of ITH as a prognostic biomarker. Originally, measurements of intra-lesion genetic diversity within the premalignant condition Barrett’s oesophagus were found to be strong predictors of cancer development risk40, and the prognostic value of the diversity signal appeared largely robust to the statistic used to quantify it80. Clonal diversity has also been found to be higher in breast cancer subtypes associated with a worse prognosis30 and is associated with worse overall survival in head and neck cancers81, acute myeloid leukaemia82, ovarian cancers37, and lung cancers11. Importantly, the prognostic value of ITH measures does not appear to be limited to measure of genetic heterogeneity, since quantification of morphological heterogeneity in the organisation of tumour and stromal cells in breast cancers83 and the ITH of positron emission tomography-computed tomography signal in lung cancers84 has also been reported to significantly correlate with outcome. These studies give support to the idea that quantification of ITH, as a measure of the carcinogenic process itself rather than a specific feature of that process, may be a universal prognostic biomarker suitable for use in all cancer types. Further work is required to determine which features of tumour biology should be assessed for heterogeneousness in order to best determine prognosis. For example, much genetic ITH may be irrelevant for tumour biology (as discussed above) and so naive genetic ITH measures may have limited prognostic value.\n\nIntriguingly, tumours that have underlying defects in DNA repair (such as mismatch repair85,86 or polymerase-epsilon87 defective tumours of the colon), which are expected to have very high levels of genetic ITH, paradoxically have very good prognoses88–90. Similarly, with an expression-based signature to quantify the degree of chromosomal instability (CIN) in breast cancers, a measure which likely correlates with ITH, the patients with the worst outcome were those whose tumours had intermediate levels of CIN, whereas patients whose tumours had very high levels of CIN had better outcomes91. One possible explanation for these data is that very high mutation rates, while generating lots of diversity that makes the tumour more evolvable, also generate lots of deleterious variants that impede clone growth. A further contributing factor to this favourable prognosis (at least in tumours with an elevated point mutation rate, such as microsatellite unstable tumours) is the elevated immunogenicity of tumours with a high mutation rate, a feature that is the result of the generation of many immunogenic neo-antigens, which stimulate the host immune system92. Effective ITH-based prognostic markers will need to address such complexities of tumour evolution to be broadly useful. We note too that existing broad molecular classifications of cancer (such as microsatellite versus chromosomally unstable cancers in the colon) can have significant prognostic value themselves, and consequently ITH-based measures may be useful only to stratify within such molecularly defined subgroups.\n\n\nIntra-tumour heterogeneity and treatment\n\nResistance to chemo- and targeted-therapy, and concomitant treatment failure, occurs in the majority of cases72,93–97. Pre-existing ITH can be (indirectly) attributed as the underlying cause of these treatment failures. Tumours, at the stage when they are treated, contain many billions of cells and it appears an almost mathematical certainty that at least a few of these cells will have evolved a therapy-resistant phenotype. Indeed, in patients with colorectal cancer treated with the anti-EGFR antibody cetuximab, empirical measurements show that KRAS-mutant clones that likely existed at undetectably low frequency prior to the initiation of therapy expand exponentially at the administration of therapy98 and similar dynamics have been observed in a variety of other malignancies, including lung99,100, leukaemia23,101, and melanoma102,103.\n\nIs ITH an insurmountable barrier to effective cancer treatment? It is clearly a major challenge, and accordingly one approach that has been suggested is to try to suppress the level of ITH within a tumour (by targeting the drivers of genetic instability) in order to provide a more homogenous tumour that may be more pliable with treatment104. An exciting but radically different approach is to try to use the presence of ITH itself in order to increase the efficacy of cancer treatment105. The approach, termed adaptive therapy, is based on the maxim that “nothing comes for free”. This is the idea that resistance to a particular therapeutic agent inevitably carries some cost to the cancer cell, so that in the absence of therapy a resistant clone is at a disadvantage and so will be outcompeted by any remaining sensitive cells. The adaptive therapy approach is therefore to pulse the drug in such a manner that when the drug is present the sensitive cell population is killed off and the resistant cell population prospers, whereas when the drug is absent the resistant population is outcompeted by the sensitive population. In theory, if the sensitive population is able to outcompete the resistant population sufficiently well, this treatment regime will mean that the tumour remains sensitised to the drug over long time frames while the size of tumour population as a whole is effectively constrained106,107. In a melanoma xenograft model, this idea appears efficacious108. Translation of this idea to the clinic would require the development of appropriate monitoring tools that indicate when to provide/withdraw treatment; this is clearly a major challenge in itself. It is important to note that the idea behind adaptive therapy is not to affect a cancer cure—indeed, the idea is predicated on the assumption that this is impossible with a single agent—but rather to extend the effectiveness of a particular agent indefinitely and in so doing make the cancer a chronic rather than fatal disease.\n\nOther approaches have been proposed that similarly attempt to steer the evolutionary response of a tumour to treatment109,110. The general idea of these innovative proposals is that it is possible to predict which phenotypes will emerge from a heterogeneous population under treatment, and so the next therapy can be applied in order to treat the emergent clone. This logic provides the rationale behind attempts to “vertically” combine targeted therapies (targeting multiple members of the same pathway) to prevent resistance to a single agent emerging because of the selective pressure the mono-agent provides for the emergence of a clone with a mutation in a gene downstream of the original drug target111,112. In general, combination therapies may also help to tackle the issue of pre-existing resistance in heterogeneous tumours, since the chance of a cell being doubly resistant to two different therapies should be proportional to the probability that the cell is resistant to any one therapy individually, and so if mono-therapy resistance is rare in the tumour cell population, then doubly resistant cells should be vanishingly rare indeed113. An emerging theme of combination therapy is the use of both targeted and non-specific therapies, such as immunotherapy. For example, a combination of BRAF and MEK inhibitors with adoptive cell transfer (ACT) immunotherapy in a BRAF-driven mouse model of melanoma has been shown to induce complete tumour regression114. However, it should be noted that combining multiple therapies may carry the cost of increased toxicity to normal tissue. Together, these data and theoretical studies suggest that therapeutic regimes will need to be personalised for the evolutionary response of an individual tumour in order to effectively tackle the problem of ITH.\n\n\nConclusions\n\nITH is an inevitable feature of all cancers and presents challenges to our understanding of tumour biology and our ability to prognose and treat cancer. But it is our opinion that these challenges are not insurmountable, and in fact understanding the processes generating ITH provides us with a window to understand the very drivers of carcinogenesis itself. From a more clinical perspective, quantification of ITH offers an exciting opportunity to improve prognostication, and exploiting rather than ignoring ITH has the potential to improve the efficacy of existing cancer therapies.", "appendix": "Competing interests\n\n\n\nThe authors declare that they have nothing to disclose. No writing assistance was used.\n\n\nGrant information\n\nLG, A-MB, and TAG were funded by Cancer Research UK.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nHirsch FR, Ottesen G, Pødenphant J, et al.: Tumor heterogeneity in lung cancer based on light microscopic features. A retrospective study of a consecutive series of 200 patients, treated surgically. Virchows Arch A Pathol Anat Histopathol. 1983; 402(2): 147–53. 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PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation" }	[ { "id": "12666", "date": "29 Feb 2016", "name": "Peter Dirks", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12667", "date": "29 Feb 2016", "name": "James Brenton", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12668", "date": "29 Feb 2016", "name": "Christine Sers", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12669", "date": "29 Feb 2016", "name": "David Basanta", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12670", "date": "29 Feb 2016", "name": "Kornelia Polyak", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-238
https://f1000research.com/articles/4-1378/v1	30 Nov 15	{ "type": "Research Article", "title": "Metabarcoding-based fungal diversity on coarse and fine particulate organic matter in a first-order stream in Nova Scotia, Canada", "authors": [ "Christian Wurzbacher", "Ivan J. Grimmett", "Felix Bärlocher", "Ivan J. Grimmett" ], "abstract": "Most streams receive substantial inputs of allochthonous organic material in the form of leaves and twigs (CPOM, coarse particulate organic matter). Mechanical and biological processing converts this into fine particulate organic matter (FPOM). Other sources of particles include flocculated dissolved matter and soil particles. Fungi are known to play a role in the CPOM conversion process, but the taxonomic affiliations of these fungi remain poorly studied. The present study seeks to shed light on the composition of fungal communities on FPOM and CPOM as assessed in a natural stream in Nova Scotia, Canada. Maple leaves were exposed in a stream for four weeks and their fungal community evaluated through pyrosequencing. Over the same period, four FPOM size fractions were collected by filtration and assessed. Particles had much lower ergosterol contents than leaves, suggesting major differences in the extent of fungal colonization. Pyrosequencing documented a total of 821 fungal operational taxonomic units (OTU), of which 726 were exclusive to particles and 47 to leaf samples. Characterizing fungal communities may shed some light on the origins and processing pathways of fine particles in streams and broadens our view of the phylogenetic composition of fungi in freshwater ecosystems.", "keywords": [ "aquatic fungi", "stream", "pyrosequencing", "CPOM", "FPOM" ], "content": "Introduction\n\nHeadwaters are almost entirely heterotrophic – up to 99% of their energy is supplied by coarse organic matter (CPOM, diameter > 1 mm) imported from the terrestrial surroundings (e.g., twigs, branches, and leaves). These allochthonous sources are converted into fine particulate organic matter (FPOM) mechanically by the water current, by feeding activities of invertebrate shredders (both by “sloppy” feeding and by feces production due to incomplete digestion; Cummins & Klug, 1979; Shepard & Minshall, 1981; Wotton et al., 1998) and by fungal maceration (Suberkropp & Klug, 1980). Stream fungi are vitally important for energy transformation of submerged leaf litter (Baldy et al., 1995; Gessner & Chauvet, 1994; Gulis & Suberkropp, 2003; Hieber & Gessner, 2002). FPOM may also be blown in or washed in from adjacent forest soils, or originate from sloughed-off algal biofilms, consist of plant spores and pollen (Czeczuga & Orlowska, 2001), or be produced by flocculation of DOM, with or without microbial participation (Lush & Hynes, 1973; Wotton, 1990). Due to the many biological processes involving CPOM and FPOM, bacteria and fungal spores will also contribute to the pool of stream FPOM (Bärlocher & Brendelberger, 2004; Callisto & Graça, 2013; Edwards & Meyer, 1987; Gleason et al., 2009). FPOM is one of the major components of stream ecosystems, and entire groups of organisms, such as the filter feeding guild, depend on it (Callisto & Graça, 2013; Wallace & Merritt, 1980).\n\nHowever, very little is known about FPOM associated microbial communities. Fine particles, regardless of their origin, are continually colonized and transformed by microorganisms. Due to resource limitation on small particles, we can assume that the biomass of mycelial fungi, as measured by ergosterol concentrations (Callisto & Graça, 2013; Findlay et al., 2002), will be low and that zoosporic and/or unicellular fungi will be more prominent due to their adaptations to small substrates such as algae and pollen (Gleason et al., 2008). Previous studies have shown that ascomycetous hyphomycetes are dominant stream dwelling fungi on leaf-litter (e.g. Bärlocher, 1990; Duarte et al., 2015). Some of these leaf-litter fungi survive passage through the gut of leaf-eating amphipods (Bärlocher, 1981; Sridhar et al., 2011), and DNA from both ascomycetes and chytridiomycetes is present in fecal particles (Sridhar et al., 2011).\n\nThe present paper seeks to examine the fungal community on collected stream FPOM and whether it is possible to use it as a sum parameter for various fungal processes in the stream ecosystem. For this feasibility study we collected three FPOM size fractions and compared them with four weeks old submerged leaf-litter. We measured ergosterol as a biomass indicator of Dikarya and employed a metabarcoding approach in order to classify the fungal community of stream organic matter (OM).\n\n\nMethods\n\nThe field experiment was conducted in Boss Brook, a small stream in Fenwick, Nova Scotia, Canada (45° 43.00'N, 64° 09.56'W) (Nikolcheva et al., 2003). This first-order stream runs through a mixed forest dominated by white birch (Betula papyrifera Marsh), several maple species (Acer rubrum L., Acer saccharum Marsh., Acer spicatum Lam.), and white spruce (Picea glauca [Moench] Voss). The stream bed consists of stones and gravel. At the sampling site, the stream is 2 to 3 m wide and 20 to 50 cm deep (Grimmett et al., 2012). On three dates (27 September, 25 October, 7 November 2011), 100 l of stream water were passed through a stack of metal filters, yielding 4 FPOM fractions (fraction 1: 2–1 mm; fraction 2: 1–0.5 mm; fraction 3: 0.5–0.25 mm, fraction 4: 0.25–0.020 mm). Most material was recovered in the lowest size fraction (4) and no material was recovered in fraction 1 (Dataset 1). Samples were lyophilized and weighed. Additional samples for ergosterol measurements were collected on 12 November. These were freeze-dried and stored in methanol/potassium hydroxide (3 × 15 mg in 2 ml each at – 20°C; Nikolcheva et al., 2003, Dataset 2). Samples of different dates were combined for pyrosequencing in order to adjust for the temporal variation and to increase the resolution, resulting in one sample each for size fractions 2 to 4. In parallel, senescent leaves from individual trees (Maple: Acer platanoides) were incubated as leaf discs (15 cm) in duplicate bags in the stream for four weeks (11 October to 9 November, 2011) to evaluate fungal communities on CPOM (procedures as in Grimmett et al., 2012). As an (aquatic) outgroup we incubated leaves from a non-native tree (European beech, Fagus sylvatica) in the littoral zone of a lentic system (Lake Utopia, near St. George, NB, Canada; 45° 10.18'N 66° 47.67'W) for two weeks. The goal was to obtain an indication if substrate type (leaf vs. fine particles) may be more important than species (maple vs. beech) or habitat (lotic vs. lentic). All leaf samples were stored at -20°C until DNA extraction. In total we sequenced 6 samples consisting of two replicates (Maple I and Maple II) from the leaf bags, one lake-derived beech leaf bag sample, and one sample per stream-particle sample.\n\nDNA from freeze-dried particle fractions and frozen leaf samples was extracted with the PowerSoil MoBio Kit as per the manufacturer's instructions (leaves were first cut into smaller pieces with a sterile scalpel). Amplicon PCR was performed using the barcoded 18S primers nu-SSU-0817 and nu-SSU-1536 of Borneman & Hartin (2000), and AccuPrime High Fidelity Polymerase (Life Technologies # 12337016) in a two-step PCR for 32 cycles (94°C for 1 min and 60°C for 4 min) with an initial denaturation for 5 min; BSA was added at a final concentration of 0.9 µg µl-1. Amplicons were prepared according to the Lib-L protocol (454 Life Sciences, Roche) and sequenced by a benchtop GS Junior System (454 Life Sciences, Roche). Raw data were processed by Mothur (version 1.26, Schloss et al., 2011), following recommendations for standard operating procedure (http://www.mothur.org/wiki/Schloss_SOP, accessed 7/2012), implementing denoising, trimming, alignment, filtering, chimera removal, classification, and preclustering steps against the eukaryotic reference database provided by Mothur. OTUs were calculated on a 97% basis of a final alignment with a median length of 279 nt and statistics (diversity estimates and similarities) were done with a random submatrix normalized to the lowest number of reads. After the filtering process and removal of chimeric sequences, there were 10,000–22,000 reads per sample, resulting in a sampling coverage of > 99%. The OTU table (Dataset 3) for those reads was exported to R (version 2.15.1) for cluster analysis with the second Kulczynski similarity index (http://cran.r-project.org/). In addition, representative OTUs of the associated FASTA file were realigned with SINA (version 1.2.11, Pruesse et al., 2012) and imported into the SILVA SSU reference database version 111 (http://www.arb-silva.de/). The representative sequences from all OTUs were added to the SILVA reference database by the parsimony option activating the eukaryotic positional variability filter implemented in ARB (version 5.5, Ludwig et al., 2004). The resulting tree (i.e. the top 100 subtree) was exported and processed by FigTree (version 1.4, http://tree.bio.ed.ac.uk/software/figtree/). The raw sequences were deposited in the European Nucleotide Archive (ENA; accession number PRJEB10809).\n\n\nResults\n\nAverage stream FPOM concentration of the three sampling dates was 2.7 mg l-1. Distribution among the various size fractions is summarized in Table 1. Due to fluctuating water flow, there was a high temporal variation in the amount of recovered particles. On average the smallest size fraction (250 µm – 20 µm) was the most abundant. Ergosterol concentrations decreased with lower particle size (Table 1). It was highest on maple leaves recovered from Boss Brook and also higher on our lentic outgroup: beech leaves from Lake Utopia.\n\nErgosterol values were evaluated by ANOVA (p < 0.0001), followed by Tukey-Kramer. Averages with same letter are not significantly different (p > 0.05). Based on Dataset 1 and Dataset 2.\n\nThe DNA sequences were assigned to 821 fungal OTUs. Of these, 726 were detected exclusively in the particle fractions, and 47 (out of 95 OTUs detected on leaves) were restricted to leaf samples. The particles shared 130 OTUs (with an extrapolated shared species Chao index of 215.5). The taxon richness of the particles was an order of magnitude higher than the one of maple leaf samples and the inverse Simpson index pointed to a more even and diverse community structure on particles than on leaves (Table 2). This is further reflected in the rank abundance curves of the two substrate types (Figure 1). Correspondingly, the analysis of fungal communities separates leaves from particles with less than 40% similarity (Figure 2). When we looked at the most prominent 100 OTUs from stream particles, which accounted for 96% of all sequences (Figure 1), we found representatives of most fungal phyla including yeast lineages (e.g. Taphrinaceae and Saccharomycotina), Basidiomycota, and the phyla Chytridiomycota and Cryptomycota (Figure 3). The fungal diversity was dominated by several classes of Pezizomycotina (Ascomycota).\n\nData based on a Kulczynski similarity matrix, clustered with UPGMA (average) method (based on Dataset 3).\n\nNumbers of retrieved sequences per OTU are written at the outer rim; asterisks mark taxa that were also recovered on leaf litter. Branches without numbers are reference sequences (SILVA) (based on Dataset 3).\n\nIn order to trace back fungal taxa derived from leaf-litter decomposition (marked with asterisks in Figure 3), we looked at the most abundant OTUs on the maple leaves incubated in the stream (Table 3). OTU 1 (Pezizomycotina) was the most abundant OTU in all samples. On average between 62.0 – 72.1% of the taxa found on each of the FPOM fractions were also present on submerged leaf litter. To get rough estimates of alternative origins and functions of the fungal communities on stream particles, we split them into different categories: potential soil fungi (with Agaricomycetes as proxy) accounted for 4.8 – 12.3%, yeast-like fungi made up 5.3 – 6.8%, and non-Dikarya fungal lineages ranged between 0.8 – 1.6%.\n\nThe table is sorted after the most abundant OTUs on maple leaves in descending order. (n.d. = not detected, based on Dataset 3).\n\n\nDiscussion\n\nIn this study we focussed on stream particles and we were especially interested in the fungal phyla we may find on them. Thus we applied a conservative marker gene, which is especially efficient at resolving the basal branches of Fungi (Mohamed & Martiny, 2011). With this we could successfully detect a broad spectrum of fungal phyla on FPOM, including Chytridiomycota sequences and taxa belonging to the newly described group of Cryptomycota (Jones et al., 2011). Chytridiomycota have been documented on leaf litter in freshwater streams before (Bärlocher et al., 2011; Marano et al., 2011; Nikolcheva & Bärlocher, 2004), but, to our knowledge, this is the first study to document Cryptomycota in streams (Jones et al., 2011). Cryptomycota are assumed to be parasitic on various organisms including fungi (Gleason et al., 2012), however, some evidence also points to a saprobic life style (Wurzbacher et al., 2014). Their ecological role in streams needs to be further elucidated, especially since they have the potential for mycoparasitism. We did not find typical trichomycete sequences (e.g. Lichtwardt, 1972), which would have pointed to a gut passage of particles through filter-feeders. Possibly, the sampling sites had an insufficient number of filter feeders or too few gut fungi on fecal pellets to allow detection using our methods.\n\nThe high diversity of fungi on stream particles stands in contrast to the very low diversity on leaf-litter. It is likely that the dominant OTU 1 comprises several prominent aquatic hyphomycete species, since these rarely differ in their nuclear SSU sequence (Belliveau & Bärlocher, 2005, see also Tedersoo et al., 2015 for general limitations of SSU for Dikarya). In general we think that the high diversity on particles reflects their multiple origins and histories. The fact that those few leaf-litter taxa were also abundant in the stream particles points to leaf-litter as one important particle origin. For example, if the stream is dominated by particles washed in from the forest we would have expected Basidiomycota to dominate (Tedersoo et al., 2014). They accounted for 72% in Lim et al. (2010) and for ≥ 60% in Shi et al. (2014) with Agaricomycetes as the most common class, a much larger proportion than on leaves or particles in the current study (4.8 – 12.3%). However, it is also conceivable that soil particles, upon immersion in a stream, undergo further processing during which Basidiomycota are rapidly replaced by indigenous stream fungi. The transport and age of FPOM and its distribution among various size classes is highly variable and strongly depends on hydrological fluctuations throughout the seasons (Bilby & Likens, 1979; Thomas et al., 2001). In other words, it is not possible to deduce the origin of the stream FPOM by looking at taxonomic composition of its mycoflora. But the high fungal diversity on stream particles points to the interaction of various stream processes. In this context it is interesting to compare our findings with an arctic study which focussed on unfractionated water samples (Crump et al., 2012) that showed that only a minor fraction of eukaryotic microorganisms (< 10%) in a first order stream originated from the soil, while the majority seemed to be indigenous. Interestingly, the situation was the reverse for prokaryotes, which were predominantly washed in from soil (Crump et al., 2012).\n\nIn our study, ergosterol concentration decreased in smaller particles, pointing to reduced biomass of living fungi derived from e.g. leaf-litter. Still, fungal OTUs found on submerged CPOM (leaf-litter) dominate the sequences on all particle size classes. This suggests that most fungal taxa were reduced non-selectively during the processing of CPOM to FPOM, or that the fungal cells were degraded (as suggested by the decline of ergosterol) but their DNA remained largely intact and attached to the particles as environmental DNA. Such bound DNA can remain stable for extended periods of time (Guggenberger & Kaiser, 2003; Nguyen & Elimelech, 2007) and it is known that extracellular DNA occurs in considerable quantities in aquatic systems and sediments (summarized in Pietramellara et al., 2009).\n\nIn order to test the hypothesis that the fungal community of FPOM is indeed a function of present stream processes, the storage potential of FPOM has to be defined accurately by investigating the fungal taxa turnover on particles of known origin and composition incubated in a stream. Supplementing these approaches with ribosomal RNA will allow us to control for the proportion of environmental DNA. Much of the biological processing of FPOM in streams is still unclear (Tank et al., 2010), and tracking their DNA levels and diversity might shed some light on their origin, history and in-stream transport.\n\n\nConclusions\n\nWe successfully looked at the broad phylogenetic diversity of stream FPOM (> 20 µm), which was much higher than on leaf-litter and included members of novel groups (Cryptomycota). The most abundant operational taxonomic units on particles were identical to taxa on submerged decomposing leaf litter. We documented distinct differences in ergosterol content between particle sizes, which points to the near absence of living Dikarya mycelium on smaller stream particles. More conclusive evidence will be required to unravel the relative effects of origin (e.g., leaves decomposing in the stream vs. soil particles vs. algal particles) and processing or ageing of particles including DNA storage within the fungal community. Combining this information should allow us to more fully document various stream processes initiated by fungal organism.\n\n\nData availability\n\nRaw sequence data for the samples reported here can be found in the European Nucleotide Archive (http://www.ebi.ac.uk/ena), under accession PRJEB10809.\n\nF1000Research: Dataset 1. Amount of particles in the stream, 10.5256/f1000research.7359.d107489 (Wurzbacher et al., 2015a).\n\nF1000Research: Dataset 2. Ergosterol content of particle size fractions, 10.5256/f1000research.7359.d107490 (Wurzbacher et al., 2015b).\n\nF1000Research: Dataset 3. OTU matrix including fasta sequences, 10.5256/f1000research.7359.d107491 (Wurzbacher et al., 2015c).", "appendix": "Author contributions\n\n\n\nCW, IG and FB conceived the study. CW designed the experiments. CW and IG carried out the research. CW and FB prepared the first draft of the manuscript. IG contributed to the experimental design and preparation of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis research was supported by an NSERC Discovery Grant to FB.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nWe acknowledge Keegan Smith for field assistance, and Natalie Donaher and Miranda Corkum for technical assistance. We gratefully acknowledge Hans-Peter Grossart and Nicola Wannicke for helpful discussions. This is publication 009 of the Berlin Center for Genomics in Biodiversity Research.\n\n\nReferences\n\nBaldy V, Gessner MO, Chauvet E: Bacteria, fungi and the breakdown of leaf litter in a large river. Oikos. 1995; 74(1): 93–102. Publisher Full Text\n\nBärlocher F: Fungi on the food and in the faeces of Gammarus pulex. T Brit Mycol Soc. 1981; 76(1): 160–165. 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Biol Fertil Soils. 2009; 45(3): 219–235. Publisher Full Text\n\nPruesse E, Peplies J, Glöckner FO: SINA: Accurate high-throughput multiple sequence alignment of ribosomal RNA genes. Bioinformatics. 2012; 28(14): 1823–1829. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchloss PD, Gevers D, Westcott SL: Reducing the effects of PCR amplification and sequencing artifacts on 16S rRNA-based studies. PLoS One. 2011; 6(12): e27310. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShepard RB, Minshall GW: Nutritional value of lotic insect feces compared with allochthonous materials. Archiv für Hydrobiologie. 1981; 90(4): 467–488. Reference Source\n\nShi LL, Mortimer PE, Ferry Slik JW, et al.: Variation in forest soil fungal diversity along a latitudinal gradient. Fungal Divers. 2014; 64(1): 305–315. Publisher Full Text\n\nSridhar KR, Beaton M, Bärlocher F: Fungal propagules and DNA in feces of two detritus-feeding amphipods. Microb Ecol. 2011; 61(1): 31–40. PubMed Abstract \| Publisher Full Text\n\nSuberkropp K, Klug MJ: The maceration of deciduous leaf litter by aquatic hyphomycetes. Can J Bot. 1980; 58(9): 1025–1031. Publisher Full Text\n\nTank JL, Rosi-Marshall EJ, Griffiths NA, et al.: A review of allochthonous organic matter dynamics and metabolism in streams. J North Am Benthol Soc. 2010; 29(1): 118–146. Publisher Full Text\n\nTedersoo L, Bahram M, Põlme S, et al.: Fungal biogeography. Global diversity and geography of soil fungi. Science. 2014; 346(6213): 1256688. PubMed Abstract \| Publisher Full Text\n\nTedersoo L, Anslan S, Bahram M, et al.: Shotgun metagenomes and multiple primer pair-barcode combinations of amplicons reveal biases in metabarcoding analyses of fungi. MycoKeys. 2015; 10: 1–43. Publisher Full Text\n\nThomas SA, Newbold JD, Monaghan MT, et al.: The influence of particle size on seston deposition in streams. Limnol Oceanogr. 2001; 46(6): 15–24. Publisher Full Text\n\nWallace JB, Merritt RW: Filter-feeding ecology of aquatic insects. Annu Rev Entomol. 1980; 25: 103–132. Publisher Full Text\n\nWotton RS: The Biology of Particles in Aquatic Systems. CRC Press, 1990; 1–336. Reference Source\n\nWotton RS, Malmquist B, Muotka T, et al.: Fecal pellets from a dense aggregation of suspension-feeders in a stream: an example of ecosystem engineering. Limnol Oceanogr. 1998; 43(4): 719–725. Publisher Full Text\n\nWurzbacher C, Rösel S, Rychła A, et al.: Importance of saprotrophic freshwater fungi for pollen degradation. PLoS One. 2014; 9(4): e94643. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWurzbacher C, Grimmett IJ, Bärlocher F: Dataset 1 in: Metabarcoding-based fungal diversity on coarse and fine particulate organic matter in a first-order stream in Nova Scotia, Canada. F1000Research. 2015a. Data Source\n\nWurzbacher C, Grimmett IJ, Bärlocher F: Dataset 2 in: Metabarcoding-based fungal diversity on coarse and fine particulate organic matter in a first-order stream in Nova Scotia, Canada. F1000Research. 2015b. Data Source\n\nWurzbacher C, Grimmett IJ, Bärlocher F: Dataset 3 in: Metabarcoding-based fungal diversity on coarse and fine particulate organic matter in a first-order stream in Nova Scotia, Canada. F1000Research. 2015c. Data Source" }	[ { "id": "12182", "date": "03 Feb 2016", "name": "Belle Damadora Shenoy", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript is well-written, and will be acceptable after a few minor suggestions are incorporated: The abstract should contain more information on the results of the paper. I would rather like to see (in the abstract) how much light your pyrosequencing has shed on the origins and processing pathways of fine particles. In the discussion section, the authors write “[…] to our knowledge, this is the first study to document Cryptomycota in streams (Jones et al., 2011).” – the authors could highlight recent reports on Cryptomycota from fresh water bodies. In the conclusions, the authors write “In other words, it is not possible to deduce the origin of the stream FPOM by looking at taxonomic composition of its mycoflora.”. This is a possible contradiction with the last sentences of the abstract and discussion sections?These suggestions are also available as an annotated PDF.", "responses": [ { "c_id": "1825", "date": "26 Feb 2016", "name": "Christian Wurzbacher", "role": "Author Response", "response": "Thank you very much for your review. We amended your earlier suggestions in Version 2 of the article:\"In this version we followed the suggestions of the one of the referee reports by (i) adding more results to the abstract, (ii) giving another reference for recent discussions on Cryptomycota in freshwater systems, and by (iii) consistently stating that it may not be possible to deduce the origin of the particle by analysing its fungal community.\"With kind regards" } ] }, { "id": "12451", "date": "15 Feb 2016", "name": "Huzefa A Raja", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 well written manuscript. I do not have much to comment, except that this is a well thought out experimental study. It is very interesting to note that a larger number of OTUs were obtained from FPOM, whereas, the OTUs from CPOM (leaf litter) were fewer in number. Finding members of Cryptomycota in streams for the first time was interesting. I would have liked to see the ITS region sequenced, so we could get a better understanding of the species-level identification.", "responses": [] } ]	1	https://f1000research.com/articles/4-1378
https://f1000research.com/articles/5-224/v1	26 Feb 16	{ "type": "Review", "title": "IL-21 Signaling in Immunity", "authors": [ "Warren J. Leonard", "Chi-Keung Wan", "Chi-Keung Wan" ], "abstract": "IL-21 is a type I cytokine produced by T cells and natural killer T cells that has pleiotropic actions on a wide range of immune and non-immune cell types. Since its discovery in 2000, extensive studies on the biological actions of IL-21 have been performed in vitro and in vivo. Recent reports describing patients with primary immunodeficiency caused by mutations of IL21 or IL21R have further deepened our knowledge of the role of this cytokine in host defense. Elucidation of the molecular mechanisms that mediate IL-21’s actions has provided the rationale for targeting IL-21 and IL-21 downstream mediators for therapeutic purposes. The use of next-generation sequencing technology has provided further insights into the complexity of IL-21 signaling and has identified transcription factors and co-factors involved in mediating the actions of this cytokine. In this review, we discuss recent advances in the biology and signaling of IL-21 and how this knowledge can be potentially translated into clinical settings.", "keywords": [ "cytokine", "IL-21", "Immunosuppression", "cancer immunotherapy", "B-cell differentiation", "T-cell differentiation" ], "content": "Introduction\n\nIL-21 is a pleiotropic type I cytokine that is produced mainly by T cells and natural killer T (NKT) cells. This cytokine has diverse effects on a broad range of cell types including, but not limited to, CD4+ and CD8+ T cells, B cells, macrophages, monocytes, and dendritic cells (DCs)1 (Figure 1). The functional receptor for IL-21 is composed of the IL-21 receptor (IL-21R) and the common cytokine receptor γ chain (γc), which is also a subunit of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15. Mutations of γc in humans result in X-linked severe combined immunodeficiency (XSCID), a disease characterized by the absence of T cells and natural killer (NK) cells, and with B cells that are normal in number but non-functional2. It is now clear that defective IL-21 signaling substantially explains the defective B-cell function in this disease3,4. In the past few years, the use of next-generation sequencing technology, particularly chromatin immunoprecipitation coupled with DNA sequencing (ChIP-Seq) and RNA-Seq, has provided insights into the complexity of cell type-specific IL-21-mediated signaling and helped to identify the transcription factors and co-factors involved5. The pathogenic role of IL-21 in various types of autoimmune diseases is supported by the use of animal models, clinical reports, and genome-wide association studies (GWAS)1. Moreover, reports describing patients with primary immunodeficiency caused by IL21 or IL21R mutations underscore the critical role of IL-21 in host defense in vivo in humans6–9. Knowledge of the biological functions of IL-21 has led to clinical trials using this cytokine alone or in combination with other agents in treating metastatic cancers, and blocking antibodies to IL-21R are now being evaluated in clinical trials for the treatment of autoimmune diseases. Interestingly, discovery of the immunosuppressive actions of IL-21 suggests that this cytokine is a “double-edged sword” – IL-21 stimulation may lead to either the induction or suppression of immune responses, so that both stimulatory and suppressive effects of IL-21 must be considered during the clinical use of IL-21-related immunotherapeutic agents. The biological effects of IL-21 are also influenced by the presence of other cytokines or signaling molecules in the microenvironment. Here, we review recent advances in our understanding of the biology and signaling of IL-21 and potential clinical applications.\n\nIL-21 is produced mainly by CD4+ T cells and NKT cells (see bold arrows), but it is also produced by CD8+ T cells. IL-21 acts on both lymphoid and myeloid populations and can positively or negatively regulate immune responses depending on the context. The text in red indicates biological actions that make IL-21 a potential anti-cancer agent: it enhances the cytotoxic actions of CD8+ T cells and NK cells, induces apoptosis of B cell lymphoma cells, and promotes the M2 to M1 transition of the tumor-associated macrophages. The text in blue indicates actions of IL-21 that may contribute to autoimmune diseases: differentiation of Tfh and Th17 cells, inhibition of Treg generation, and the production of auto-antibodies. Thus, blocking IL-21 signaling has promising therapeutic potential.\n\n\nIL-21-activated STAT3 forms cell type-specific complexes for signaling\n\nAnalogous to other γc cytokines, IL-21 transduces molecular signals substantially via the Janus kinase and Signal Transducer and Activator of Transcription (JAK-STAT), phosphoinositide 3-kinase (PI3K), and mitogen-activated protein kinase (MAPK) pathways10. IL-21 induces strong and sustained activation of STAT3, which is critical for its effects on B-cell and T-cell differentiation1. The clinical significance of STAT3 in IL-21-mediated signaling has been confirmed in patients with STAT3 mutations11–15. Studies have identified additional transcriptional factors and co-factors involved in IL-21-mediated signaling, with some of them forming complexes with STAT35. In addition to STAT3, IL-21-induced T-helper (Th) 17 cell differentiation requires interferon regulatory 4 (IRF4), with Irf4-deficient CD4+ T cells having defects in IL-17 production after stimulation with IL-21 and TGF-β16. ChIP-Seq analysis in both B cells and CD4+ T cells has revealed global cooperative activity of the IL-21-activated STAT3 with IRF4, with most regions with STAT3 binding activity also binding IRF4, and moreover IL-21-mediated, STAT3-dependent gene expression is diminished in the absence of IRF417. IRF4 itself weakly binds to the DNA due to the presence of a carboxy-terminal auto-inhibitory domain, and in B cells, cooperative binding of IRF4 with ETS family proteins PU.1 or SPIB is known to increase the binding affinity of IRF4, resulting in the use of ETS-IRF4 composite elements, or EICEs. However, expression of PU.1 and SPIB is low in CD4+ T cells, leading to the unexpected discovery that, whereas B cells use EICEs, in T cells IRF4 cooperates with AP1 family proteins BATF and JUN and utilizes AP1-IRF4 composite elements (AICEs)18–20. Moreover, one study showed that cooperative activity of STAT3 and the aryl hydrocarbon receptor (AhR) is required for the expression of IL-22 in CD4+ T cells21, indicating that the protein complexes activated by IL-21 likely involve additional proteins. These studies suggest that IL-21-mediated gene regulation often requires IRF4 in B and T cells, but IRF4 in T cells additionally complexes with AP-1 family proteins to regulate expression of certain genes, and perhaps this explains some T-cell-specific actions of IL-21 (Figure 2).\n\nIL-21 activates JAK-STAT, PI 3-kinase (PI3K), and MAP kinase (MAPK) pathways. STAT3 plays a major role in the biological actions of IL-21, but STAT1 also contributes to IL-21-regulated gene expression. Opposing actions of STAT1 and STAT3 are important for fine-tuning IL-21’s functions. The importance of IL-21-activated STAT5 is not known. MAPK and PI3K pathways contribute to the proliferative effect of IL-21. In T cells, after IL-21 stimulation, optimal STAT3-mediated gene regulation requires functional cooperation with IRF4, which binds together with AP-1 family proteins (predominately BATF and JUN family proteins), to regulate genes containing AP1-IRF4 composite elements (AICEs). AhR can also cooperate with STAT3 for gene regulation in T cells after IL-21 stimulation. Additional transcription factors (TFs) and co-factors may also be involved.\n\nA critical role for STAT3 in IL-21 signaling was also confirmed in patients with autosomal dominant hyper-IgE syndrome (AD-HIES), which is caused by loss-of-function mutations of STAT3. Consistent with a key role of IL-21 in Th17 cell differentiation, CD4+ T cells from these patients are not able to produce IL-17 in vitro, and, together with the defective IL-6 and IL-23R signaling22,23, this helps to explain their susceptibility to recurrent infections24. Similarly, early studies showed that IL-21 together with CD40 engagement stimulates the differentiation of naïve B cells into IgG-producing plasma cells25, and it was subsequently shown that naïve B cells from AD-HIES patients are not able to differentiate into IgG- or IgA-producing cells after IL-21 stimulation in vitro15. However, the clinical manifestations in these patients cannot be explained solely by defective IL-21 signaling, as other cytokines including IL-6, IL-10, and IL-11 also strongly activate STAT3 for signal transduction26.\n\nIL-21 stimulation also leads to the activation of STAT1, at least in T cells, B cells, and conventional dendritic cells (cDCs), and recent studies have improved our understanding of the role of STAT1 in IL-21 signaling. IL-21-stimulated plasma cell generation remains intact in naïve B cells from STAT1 loss-of-function patients, but STAT1 plays a role in sustaining Ig production by differentiated memory B cells15. In addition, IL-21 can enhance the cytotoxic activity of mouse CD8+ T cells by induction of T-bet, predominantly via STAT127. Moreover, a recent study showed that ~10% of IL-21-regulated genes in pre-activated CD4+ T cells are dependent on STAT1, compared to ~40% being dependent on STAT328. Strikingly, expression of some genes including Th1 signature genes Ifng and Tbx21 by IL-21 are differentially regulated by STAT1 and STAT3, and IL-21-induced expression of IFNG and TBX21 is enhanced in CD4+ T cells from AD-HIES patients and also modestly increased in CD4+ T cells from STAT1 gain-of-function patients28. These findings suggest that an interplay between STAT1 and STAT3 may fine-tune IL-21-induced biological actions. This conclusion is also supported by the fact the STAT3 loss-of-function (AD-HIES) and STAT1 gain-of function patients share immunological phenotypes (e.g., defective IL-17 production in CD4+ T cells and impaired production of antigen-specific antibodies) and clinical manifestations (e.g., mucocutaneous candidiasis)12,24,29,30.\n\n\nBiological functions of IL-21 revealed from studies of patients with IL21R or IL21 mutations\n\nPatients with primary immunodeficiency caused by IL21R or IL21 mutations have been described6–9, and their phenotypes have provided invaluable insights into the role of IL-21 in host defense. Patients with defective IL-21 signaling suffer from recurrent pulmonary infections, and patients with IL21R mutations, but not the single described patient with IL21 mutations, additionally have cryptosporidiosis, leading to secondary cholangitis and liver disease. Infections with opportunistic pathogens may be due to the defects in both innate and adaptive immunity in these patients, as plasma cell and memory B-cell generation, as well as immunoglobulin class switching are impaired15, while CD8+ T-cell proliferation and NK -cell cytotoxicity are also diminished. Interestingly, the patient with the IL21 mutation did not have cryptosporidiosis but had early-onset inflammatory bowel disease (IBD)7.\n\n\nImmunosuppressive effects of IL-21\n\nThe early onset IBD observed in the one IL21-mutated patient, alongside chronic diarrhea in some IL21R-deficient patients, was interesting, as multiple studies using animal models showed that IL-21 promotes the pathogenesis of IBD31. However, IL-21 can also be immunosuppressive because of its ability to induce IL-10. IL-10 signaling is known to critically regulate immune homeostasis in the gut, and patients with IL1032 or IL10R33 mutations also develop severe early onset IBD. Interestingly, under Th1 priming conditions, the addition of IL-21 was shown to potently inhibit antigen-induced IL-2Rα expression and cell cycle progression of naïve CD8+ T cells in a STAT3-mediated, IL-10-dependent fashion34. Moreover, IL-27-mediated differentiation of IL-10-producing regulatory type 1 (Tr1) cells requires IL-21, c-Maf, and ICOS, with IL-21 acting as an autocrine factor to maintain Tr1 cells35. A more recent report showed that IL-21 drives human cord blood T cells into IL-10-producing Th1 cells36, suggesting that IL-21 can also exhibit immunosuppressive effects in humans. IL-21 together with CD40L induces human B cells to produce IL-10, particularly in memory B cells that have undergone immunoglobulin class switching37, showing that the induction of IL-10 by IL-21 is not restricted to T cells. In fact, IL-10-producing regulatory B cells (B10 cells) can be greatly expanded in vitro by engagement of CD40 and IL-21 receptors, and transferring these cells into mice significantly inhibits disease symptoms in experimental autoimmune encephalomyelitis (EAE), a model of human multiple sclerosis38. IL-21-derived B10 cells also express granzyme B, which degrades the T-cell receptor ζ-chain and limits T-cell proliferation39, providing an additional mechanism by which these cells can suppress immune responses.\n\nFurthermore, IL-21 can potently induce apoptosis of B cells25,40,41 and cDCs42, which may provide alternative mechanisms for its suppressive effect. Stimulation of mouse naïve B cells in vitro with IL-21 without co-stimulatory signals induces apoptosis via the induction of pro-apoptotic BIM expression40 and suppression of pro-survival BCL2 and BCLXL41. However, pre-activation of B cells with anti-CD40 and anti-IgM inhibits the apoptotic effect of IL-2125,40,41, and CD40 engagement combined with IL-21 drives B-cell differentiation to plasma cells via induction of BLIMP1 and stimulates Ig class-switching via STAT3 activation12,25,43,44. These results suggest that, analogous to IL-2-mediated activation-induced cell death in T cells45, IL-21 might help to eliminate B cells that are activated in an antigenic non-specific fashion without the cognate antigen-specific or co-stimulatory signals.\n\nIL-21 is known to inhibit the maturation and function of bone marrow-derived dendritic cells (BMDCs)46,47. It also inhibits the LPS-stimulated expression of pro-inflammatory cytokines IL-6 and IL-1β by these cells46. IL-21 can potently induce the apoptosis of cDCs via STAT3-mediated BIM induction42. The apoptosis induced by IL-21 can be prevented by GM-CSF, which activates STAT5. Interestingly, ChIP-Seq analysis shows that STAT3 and STAT5 compete for DNA binding at the Bim locus, suggesting a direct competitive effect for these STAT proteins. Further investigation of the effect of IL-21 on cDCs revealed an unexpected role of IL-21 in IL-1β expression via a NF-κB-independent, STAT3-dependent pathway, with direct STAT3 binding identified in the Il1b locus in cDCs after IL-21 stimulation48. These studies suggest that IL-21 has dual roles in DCs where it suppresses immune responses by inhibiting the maturation and actions of BMDCs and inducing apoptosis in cDCs, but promotes immune responses by inducing IL-1β in cDCs.\n\nThere are extensive data indicating that IL-21 signaling promotes the pathogenesis of autoimmune diseases1, including in animal models of type 1 diabetes49,50, systemic lupus erythematosus (SLE)51, and experimental autoimmune uveitis52. Moreover, the number of IL-21-producing CD4+ T cells is higher in patients with active SLE53 and chronic rheumatoid arthritis (RA)54, suggesting that blocking IL-21 signaling might serve to ameliorate these diseases. However, the effects of IL-21 can be complex, and IL-21 signaling in certain cell types can have protective effects as well. For example, in SLE-prone BXSB-Yaa mice, although selective ablation of IL-21R expression in B cells protects the mice from developing disease manifestations, IL-21 signaling supports the expansion of CD8+ suppressor T cells in these mice and, as a result, selective ablation of IL-21R in CD8+ T cells also promotes pathogenesis of the disease55. Also, IL-21-induced IL-22 expression in CD4+ T cells may play a protective role in the DSS-induced colitis model21. Thus, although blocking IL-21 signaling is currently under evaluation in early clinical trials for the treatment of autoimmune disease, it conceivably could have mixed effects depending on the context in individual patients.\n\n\nIL-21 is a promising immunotherapeutic agent for cancer\n\nActivation of the cytotoxic programs in NK cells and CD8+ T cells is key for cancer immunotherapy, and consequently early studies provided compelling evidence that IL-21 is a promising immunotherapeutic agent for this disease56. IL-21 promotes maturation, enhances cytotoxicity, and induces production of IFN-γ and perforin by NK cells57,58. Correspondingly, cytolytic activity induced by IL-21 significantly inhibits the growth of B16 melanoma58,59. Moreover, IL-21 together with IL-15 expands antigen-specific CD8+ T-cell numbers and their effector function, resulting in tumor regression60. In addition, cancer cells over-expressing IL-21 cannot graft to the host and are rapidly eliminated61–64, and local delivery of IL-21 into the tumor microenvironment in a breast tumor model was shown to switch tumor-associated macrophages from the M2 phenotype to a tumor-inhibiting M1 phenotype, which rapidly stimulates T cell responses65. These studies suggest that IL-21 can “rejuvenate” multiple effector cells in the tumor microenvironment and thus that this cytokine might be used alone or in combination with other therapeutic agents in a clinical setting. Indeed, clinical trials are underway, with encouraging results1. In one phase II study in which IL-21 was used as a single agent to treat patients with metastatic melanoma who had not received prior systemic therapy, a response rate of 22.5% was achieved66. Another phase 1/2 study investigated the effects of IL-21 combined with the tyrosine kinase inhibitor sorafenib for treating metastatic renal cell carcinoma, and a disease control rate of 82% was achieved67.\n\nIL-21 is known to directly induce apoptosis in certain types of lymphoma. In vitro studies showed that IL-21 potently induces apoptosis of diffuse large B-cell lymphoma68, mantle cell lymphoma69,70, and chronic lymphocytic leukemia71 cells via activation of STAT3 or STAT1, leading to the altered expression of BCL2 family proteins and the activation of caspases. Besides its direct apoptotic effect, IL-21 alone or combined with anti-CD20 monoclonal antibody (mAb) (rituximab) can also indirectly kill the IL-21-insensitive cancer cells by activating NK cell-dependent cytotoxic effects69,72. Based on these results, a phase I study combined IL-21 with rituximab for treating 19 patients with indolent B-cell malignancies, and 42% of patients obtained complete or partial responses73. Unlike IL-2, injection of high-dose IL-21 does not cause capillary leak syndrome in vivo74 and was well tolerated.\n\nAdoptive transfer of in vitro expanded tumor antigen-specific CD8+ T cells into patients is another promising anti-cancer strategy. When leukemia antigen-specific CD8+ T cells purified from an HLA-matched donor were cultured with IL-21 in vitro and then infused into a patient, the CD8+ T cells showed a long-lived memory phenotype compared to the cells not treated with IL-21. Patients receiving the IL-21-cultured cells had a marked decrease in leukemic cells and a sustained complete remission75. These results indicate that IL-21 may be a potent adjuvant for cell-based cancer immunotherapy.\n\n\nCritical role of IL-21 in chronic viral infection\n\nThe vital role of IL-21 in anti-viral immunity has been demonstrated mainly in studies using models of chronic lymphocytic choriomeningitis virus (LCMV) infection76–79. During chronic LCMV infection, IL-21 is produced by CD4+ T cells, which sustains CD8+ T cell expansion and production of IFN-γ, TNF-α, and IL-278. Correspondingly, mice lacking IL-21 or IL-21R show diminished CD8+ T cell clonal expansion, increased exhaustion, and persistent high serum viral titers76–78, indicating that IL-21 directly acts on CD8+ T cells to limit chronic viral infections. In addition, IL-21 can also indirectly activate the anti-viral activity of CD8+ T cells by suppressing the expansion of Treg cells during chronic LCMV infection79. Although the requirements for IL-21 signaling in host defense during acute viral infection are less stringent, studies using the Armstrong (acute) strain of LCMV or vaccinia virus showed that IL-21 signaling is essential for the survival of activated CD8+ T cells and generation of long-lived memory cells80,81. Moreover, IL-21 acting on B cells and CD4+ T cells is critical for generating long-lived plasma cells after infection with an acute strain of LCMV, vesicular stomatitis virus, and influenza virus, highlighting the importance of IL-21 in humoral immunity during viral infection82.\n\n\nIL-21 as a potential vaccine adjuvant\n\nThe biological actions of IL-21 on NK cells, CD8+ T cells, and B cells described above, as well as its potent anti-viral property shown in mouse models, make it an attractive candidate for use as a vaccine adjuvant. Indeed, IL-21 has been shown to play important roles in controlling disease progression after human immunodeficiency virus (HIV) infection. Serum levels of IL-21 are significantly reduced in HIV-infected patients and correlate with CD4+ T-cell counts83. Among different disease-status groups of HIV-infected patients, only the elite controllers maintain normal production of IL-21, and IL-21-producing CD4+ T cells are decreased in HIV-infected viremic patients or patients with progressive disease84,85. Interestingly, CD8+ T cells in HIV-infected patients produce IL-21, and the frequencies of these cells are closely associated with viral control86,87, suggesting that the loss of IL-21 production correlates with disease progression. Correspondingly, recent studies showed that T follicular helper (Tfh) cells, which are the major source of IL-21, are the most efficiently infected by HIV among different CD4+ T-cell subtypes88, and defective Tfh function results in impaired humoral immunity against HIV89. The potential use of IL-21 therefore has been investigated using non-human primate models. Similar to the ex vivo effects of IL-21 on NK and CD8+ T cells isolated from HIV-infected patients90,91, injecting IL-21 into simian immunodeficiency virus (SIV)-infected rhesus macaques increases cytotoxic activity and the production of granzyme B and perforin by these cells92,93. The frequencies of SIV-specific CD8+ T cells, peripheral blood CD27+ memory B cells, and serum SIV antibodies are also increased after IL-21 administration92. Intriguingly, IL-21 injection alone or in combination with anti-retroviral therapy in SIV-infected rhesus macaques leads to the restoration of intestinal Th17 cells, which is associated with reduced microbial translocation from the intestinal lumen into the systemic circulation, systemic inflammation, and morbidity93–95. Together, these studies indicate that IL-21 can be used as an adjuvant for anti-viral therapies.\n\n\nConcluding remarks\n\nIL-21 is being intensely studied, with new information emerging on its biological effects, its signaling mechanism(s), and clinical potential. Studies in patients with mutations in IL21, IL21R and STAT3 confirmed the major roles of IL-21-activated STAT3 signaling in T-cell and B-cell differentiation and also revealed roles for its STAT3-independent signaling. ChIP-Seq analysis has successfully identified protein complexes activated by IL-21, which help to explain the cell-type-specific effects of this cytokine. Because multiple cytokines including IL-6 and IL-10 also activate STAT3, it will be interesting to know whether these cytokines activate formation of the same complexes as IL-21 or whether there are cytokine-specific complex(es). As discussed above, IL-21 is a promising agent for treating cancers. Clinical trials using IL-21 as an adjuvant for cell-based cancer immunotherapy have been encouraging. In addition, clinical trials using blocking IL-21R mAb for autoimmune diseases are ongoing1. Moreover, in vitro expansion of regulatory B10 cells by IL-21 is potent in a mouse model and thus may have potential for human autoimmunity as well, an area for future research. In mouse models, IL-21 has been shown to play critical role(s) in the development of graft-versus-host disease (GVHD)96–100, and future clinical investigation of the possibility of using IL-21-blocking agents to treat GVHD is needed. Overall, the study of the biological actions and signaling mechanisms of IL-21 has provided critical basic insights and the rationale for clinical evaluation of IL-21, both in cancer and in the use of antibodies to IL-21 in autoimmunity and potentially other diseases as well.", "appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThis work was supported by the Division of Intramural Research, National Heart, Lung, and Blood Institute, NIH.\n\n\nAcknowledgments\n\nWe thank Dr Rosanne Spolski for critical comments on the manuscript.\n\n\nReferences\n\nSpolski R, Leonard WJ: Interleukin-21: a double-edged sword with therapeutic potential. Nat Rev Drug Discov. 2014; 13(5): 379–395. PubMed Abstract \| Publisher Full Text\n\nRochman Y, Spolski R, Leonard WJ: New insights into the regulation of T cells by gamma(c) family cytokines. Nat Rev Immunol. 2009; 9(7): 480–490. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSarosiek KA, Malumbres R, Nechushtan H, et al.: Novel IL-21 signaling pathway up-regulates c-Myc and induces apoptosis of diffuse large B-cell lymphomas. Blood. 2010; 115(3): 570–580. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nBhatt S, Matthews J, Parvin S, et al.: Direct and immune-mediated cytotoxicity of interleukin-21 contributes to antitumor effects in mantle cell lymphoma. Blood. 2015; 126(13): 1555–1564. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGelebart P, Zak Z, Anand M, et al.: Interleukin-21 effectively induces apoptosis in mantle cell lymphoma through a STAT1-dependent mechanism. Leukemia. 2009; 23(10): 1836–1846. PubMed Abstract \| Publisher Full Text\n\nde Totero D, Meazza R, Zupo S, et al.: Interleukin-21 receptor (IL-21R) is up-regulated by CD40 triggering and mediates proapoptotic signals in chronic lymphocytic leukemia B cells. Blood. 2006; 107(9): 3708–3715. PubMed Abstract \| Publisher Full Text\n\nGowda A, Roda J, Hussain SR, et al.: IL-21 mediates apoptosis through up-regulation of the BH3 family member BIM and enhances both direct and antibody-dependent cellular cytotoxicity in primary chronic lymphocytic leukemia cells in vitro. Blood. 2008; 111(9): 4723–4730. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTimmerman JM, Byrd JC, Andorsky DJ, et al.: A phase I dose-finding trial of recombinant interleukin-21 and rituximab in relapsed and refractory low grade B-cell lymphoproliferative disorders. Clin Cancer Res. 2012; 18(20): 5752–5760. PubMed Abstract \| Publisher Full Text\n\nSivakumar PV, Garcia R, Waggie KS, et al.: Comparison of vascular leak syndrome in mice treated with IL21 or IL2. Comp Med. 2013; 63(1): 13–21. PubMed Abstract \| Free Full Text\n\nChapuis AG, Ragnarsson GB, Nguyen HN, et al.: Transferred WT1-reactive CD8+ T cells can mediate antileukemic activity and persist in post-transplant patients. Sci Transl Med. 2013; 5(174): 174ra27. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nElsaesser H, Sauer K, Brooks DG: IL-21 is required to control chronic viral infection. Science. 2009; 324(5934): 1569–1572. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nYi JS, Du M, Zajac AJ: A vital role for interleukin-21 in the control of a chronic viral infection. Science. 2009; 324(5934): 1572–1576. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nFröhlich A, Kisielow J, Schmitz I, et al.: IL-21R on T cells is critical for sustained functionality and control of chronic viral infection. Science. 2009; 324(5934): 1576–1580. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nSchmitz I, Schneider C, Fröhlich A, et al.: IL-21 restricts virus-driven Treg cell expansion in chronic LCMV infection. PLoS Pathog. 2013; 9(5): e1003362. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNovy P, Huang X, Leonard WJ, et al.: Intrinsic IL-21 signaling is critical for CD8 T cell survival and memory formation in response to vaccinia viral infection. J Immunol. 2011; 186(5): 2729–2738. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nCui W, Liu Y, Weinstein JS, et al.: An interleukin-21-interleukin-10-STAT3 pathway is critical for functional maturation of memory CD8+ T cells. Immunity. 2011; 35(5): 792–805. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nRasheed MA, Latner DR, Aubert RD, et al.: Interleukin-21 is a critical cytokine for the generation of virus-specific long-lived plasma cells. J Virol. 2013; 87(13): 7737–7746. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIannello A, Tremblay C, Routy JP, et al.: Decreased levels of circulating IL-21 in HIV-infected AIDS patients: correlation with CD4+ T-cell counts. Viral Immunol. 2008; 21(3): 385–388. PubMed Abstract \| Publisher Full Text\n\nIannello A, Boulassel MR, Samarani S, et al.: Dynamics and consequences of IL-21 production in HIV-infected individuals: a longitudinal and cross-sectional study. J Immunol. 2010; 184(1): 114–126. PubMed Abstract \| Publisher Full Text\n\nChevalier MF, Jülg B, Pyo A, et al.: HIV-1-specific interleukin-21+ CD4+ T cell responses contribute to durable viral control through the modulation of HIV-specific CD8+ T cell function. J Virol. 2011; 85(2): 733–741. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilliams LD, Amatya N, Bansal A, et al.: Immune activation is associated with CD8 T cell interleukin-21 production in HIV-1-infected individuals. J Virol. 2014; 88(17): 10259–10263. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilliams LD, Bansal A, Sabbaj S, et al.: Interleukin-21-producing HIV-1-specific CD8 T cells are preferentially seen in elite controllers. J Virol. 2011; 85(5): 2316–2324. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nPerreau M, Savoye AL, De Crignis E, et al.: Follicular helper T cells serve as the major CD4 T cell compartment for HIV-1 infection, replication, and production. J Exp Med. 2013; 210(1): 143–156. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nCubas RA, Mudd JC, Savoye AL, et al.: Inadequate T follicular cell help impairs B cell immunity during HIV infection. Nat Med. 2013; 19(4): 494–499. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nIannello A, Boulassel MR, Samarani S, et al.: IL-21 enhances NK cell functions and survival in healthy and HIV-infected patients with minimal stimulation of viral replication. J Leukoc Biol. 2010; 87(5): 857–867. PubMed Abstract \| Publisher Full Text\n\nStrbo N, de Armas L, Liu H, et al.: IL-21 augments natural killer effector functions in chronically HIV-infected individuals. AIDS. 2008; 22(13): 1551–1560. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPallikkuth S, Rogers K, Villinger F, et al.: Interleukin-21 administration to rhesus macaques chronically infected with simian immunodeficiency virus increases cytotoxic effector molecules in T cells and NK cells and enhances B cell function without increasing immune activation or viral replication. Vaccine. 2011; 29(49): 9229–9238. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nPallikkuth S, Micci L, Ende ZS, et al.: Maintenance of intestinal Th17 cells and reduced microbial translocation in SIV-infected rhesus macaques treated with interleukin (IL)-21. PLoS Pathog. 2013; 9(7): e1003471. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nOrtiz AM, Klase ZA, DiNapoli SR, et al.: IL-21 and probiotic therapy improve Th17 frequencies, microbial translocation, and microbiome in ARV-treated, SIV-infected macaques. Mucosal Immunol. 2015. PubMed Abstract \| Publisher Full Text\n\nMicci L, Ryan ES, Fromentin R, et al.: Interleukin-21 combined with ART reduces inflammation and viral reservoir in SIV-infected macaques. J Clin Invest. 2015; 125(12): 4497–4513. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHippen KL, Bucher C, Schirm DK, et al.: Blocking IL-21 signaling ameliorates xenogeneic GVHD induced by human lymphocytes. Blood. 2012; 119(2): 619–628. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHanash AM, Kappel LW, Yim NL, et al.: Abrogation of donor T-cell IL-21 signaling leads to tissue-specific modulation of immunity and separation of GVHD from GVL. Blood. 2011; 118(2): 446–455. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBucher C, Koch L, Vogtenhuber C, et al.: IL-21 blockade reduces graft-versus-host disease mortality by supporting inducible T regulatory cell generation. Blood. 2009; 114(26): 5375–5384. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMeguro A, Ozaki K, Hatanaka K, et al.: Lack of IL-21 signal attenuates graft-versus-leukemia effect in the absence of CD8 T-cells. Bone Marrow Transplant. 2011; 46(12): 1557–1565. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMeguro A, Ozaki K, Oh I, et al.: IL-21 is critical for GVHD in a mouse model. Bone Marrow Transplant. 2010; 45(4): 723–729. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "12562", "date": "26 Feb 2016", "name": "Howard Young", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12560", "date": "26 Feb 2016", "name": "Stuart G Tangye", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12561", "date": "26 Feb 2016", "name": "Christoph Klein", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-224
https://f1000research.com/articles/4-1399/v1	04 Dec 15	{ "type": "Review", "title": "Recent advances in the elucidation of enzymatic function in natural product biosynthesis", "authors": [ "Gao-Yi Tan", "Zixin Deng", "Tiangang Liu", "Gao-Yi Tan", "Zixin Deng" ], "abstract": "With the successful production of artemisinic acid in yeast, the promising potential of synthetic biology for natural product biosynthesis is now being realized. The recent total biosynthesis of opioids in microbes is considered to be another landmark in this field. The importance and significance of enzymes in natural product biosynthetic pathways have been re-emphasized by these advancements. Therefore, the characterization and elucidation of enzymatic function in natural product biosynthesis are undoubtedly fundamental for the development of new drugs and the heterologous biosynthesis of active natural products. Here, discoveries regarding enzymatic function in natural product biosynthesis over the past year are briefly reviewed.", "keywords": [ "natural product biosynthesis", "plant-derived drugs", "combinatorial biosynthesis" ], "content": "Introduction\n\nWilliam Campbell, Satoshi Omura, and Tu Youyou jointly won the 2015 Nobel Prize in Medicine or Physiology for discovering the anti-parasite drugs avermectin and artemisinin. Natural products, obtained from bacterial, algal, fungal, plant, and animal sources, have provided humankind with powerful weapons to combat various diseases1,2. However, many active natural products, especially plant-derived drugs, have several disadvantages, such as low productivity and unstable source supplies. In 2013, artemisinic acid, a pharmaceutical precursor of artemisinin, which is a potent anti-malarial drug produced by the sweet wormwood plant Artemisia annua3, was first produced in an engineered Saccharomyces cerevisiae strain4. Semi-synthetic artemisinin is available on the market, and its production capacity is currently about 50–60 tons per year5,6. This work marks a milestone in synthetic biology. Very recently, well-known opioids have also been successfully biosynthesized in S. cerevisiae7; this is considered to be the most complicated and elaborate engineering work ever achieved in the field, and it is a remarkable landmark in the increasingly sophisticated use of synthetic biology to engineer complex metabolic pathways into microbes8.\n\nIt seems that the riddle of natural products is being gradually solved, as targeted, complex natural products can be obtained from engineered microbial hosts. However, when we examine these cases, it is obvious that the identification and characterization of key enzymes involved in the metabolic pathways of natural products might ultimately promote tremendous innovation and creativity. We can still use the story of artemisinin as an example. More than a decade ago, the amorpha-4,11-diene synthase (ADS) gene was cloned and identified in A. annua9. Afterward, Martin et al. introduced this gene into Escherichia coli, which initially endowed the bacteria with the ability to produce amorphadiene, the sesquiterpene olefin precursor to artemisinin10. Subsequently, by applying amorphadiene synthase and a novel cytochrome P450 monooxygenase (CYP71AV1) from A. annua, artemisinic acid can be efficiently produced from amorpha-4,11-diene in engineered S. cerevisiae via a three-step oxidation process11,12. Finally, Paddon et al. constructed the complete biosynthetic pathway of artemisinic acid by using A. annua artemisinic aldehyde dehydrogenase (ALDH1) and cytochrome CYB5, which provide a highly efficient biosynthetic route for artemisinic acid production, as the artemisinic acid titers can reach 25 g/L4.\n\nConsequently, step-by-step pathway exploration and engineering led to the discovery and application of new enzymes. Therefore, the characterization and elucidation of enzymes in natural product biosynthesis have become fundamental in the development of new drugs and the heterologous biosynthesis of active natural products. Here, we briefly review progress in the elucidation of enzymatic function in natural product biosynthesis over the past year.\n\n\nIdentification of the enzymes DRS and DRR remove the last stumbling block in the complete synthesis of opioids\n\nThe secondary metabolites of higher plants include diverse chemicals, such as isoprenoids, phenolic compounds, and alkaloids. Among these natural products, alkaloids are very important medicines. Benzylisoquinoline alkaloids (BIAs) constitute a large and structurally diverse family of pharmaceutical alkaloids that contains 2,500 defined structures. BIAs, such as the well-known analgesic compounds morphine and codeine, and the antibacterial agents berberine, palmatine, and magnoflorine, are synthesized from tyrosine by members of the Papaveraceae and Berberidaceae plant families, as well as many others13. Among these compounds, opioids are the primary drugs used for pain management and palliative care. Natural opiates, such as codeine and morphine, and semi-synthetic opioids, such as hydrocodone, hydromorphone, and oxycodone, are derived from the opium poppy (Papaver somniferum). Because of the low productivity and susceptibility of industrial poppy farming to environmental factors, the opioid supply cannot meet licit medical demand14. Moreover, the chemical synthesis of these complex compounds is uneconomical because of their complex structures.\n\n(S)-reticuline (Figure 1) is the major branch-point intermediate in the biosynthesis of BIAs. However, only (R)-isomer of reticuline can be converted to opioids. Before the discovery of the enzyme that has the ability to convert (S)-reticuline to (R)-reticuline, many efforts over the past decade were made to engineer microbes to produce BIAs downstream of (S)-reticuline. By combining microbial and plant enzymes, Minami et al. successfully synthesized (S)-reticuline and its downstream products, magnoflorine and scoulerine, from dopamine in E. coli15. In the same year, Hawkins and Smolke reported that by feeding chemically synthesized (R,S)-norlaudanosoline as a starting substrate, (R,S)-reticuline could be produced in engineered yeast, and that by using the human P450 enzyme CYP2D6 in this pathway, (R)-reticuline can be specifically converted to the morphinan alkaloid salutaridine16. As noted in these studies, the starting substrates dopamine and (R, S)-norlaudanosoline are intermediate products in the BIA metabolic pathway. Years ago, Nakagawa et al. developed an E. coli fermentation system that yields (S)-reticuline from simple carbon sources17. Very recently, DeLoache et al. demonstrated the production of the key BIA intermediate (S)-reticuline from glucose in S. cerevisiae18. Thus, in microbes, the major branch-point intermediate (S)-reticuline in the BIA biosynthetic pathway can be efficiently synthesized from a simple carbon source, such as glucose.\n\nDRR, 1,2-dehydroreticuline reductase; DRS, 1,2-dehydroreticuline synthase.\n\nAdditionally, many studies have focused on the downstream (R)-reticuline metabolic pathway. In the opium poppy, the opioid compound thebaine can be converted to codeine, morphine, and so on, and can be produced by the N-demethylation of (R)-reticuline. Moreover, Thodey et al. recently reported that by engineering yeast to express heterologous genes from the plant P. somniferum and the bacterium Pseudomonas putida M10, the starting substrate thebaine can be converted to several types of opioids, such as codeine, morphine, hydromorphone, hydrocodone, and oxycodone19.\n\nIt is obvious that the last stumbling block in the total synthesis of opioids in microbes is the gap between (S)-reticuline and (R)-reticuline. A few months ago in a study in Science, a metabolite analysis of mutant opium poppy plants and heterologous protein expression experiments demonstrated that the P450 module is responsible for the conversion of (S)-reticuline to 1,2-dehydroreticuline, but that the oxidoreductase module converts 1,2-dehydroreticuline to (R)-reticuline rather than functioning as a P450 redox partner20. Subsequently, by using a plant transcriptome database and cloned genes from P. somniferum, another group identified candidate enzymes 1,2-dehydroreticuline synthase (DRS) and 1,2-dehydroreticuline reductase (DRR), which convert (S)-reticuline to 1,2-dehydroreticuline and then to (R)-reticuline21. The identification of DRS and DRR theoretically paves the way for the total biosynthesis of opioids in microbes. Several weeks ago, the total biosynthesis of opioids in yeast was completed by Smolke’s group7. In this landmark work, more than 20 enzymes derived from rodent, plants, bacteria, and yeast sources, were successfully expressed in engineered yeast (Figure 1), which ultimately realized the complete biosynthesis of opioids from sugar7. The success of microbial total biosynthesis of opioids reveals the significant functions of enzymes in recent drug development applications, as well as in the field of synthetic biology.\n\n\nStructural elucidation of key biosynthases facilitates drug development by combinatorial biosynthesis\n\nThe polyketides constitute a broad class of structurally complex, bioactive natural products that have many important therapeutic applications, such as antibiotics, anti-fungals, anti-parasitics, anti-cancer agents, and immunosuppressants. However, the emergence and spread of antibiotic resistance are gradually becoming global public health problems, which have intensified the efforts toward new drug discovery and combinatorial biosyntheses of bioactive molecules to broaden the spectrum of antibacterial agents22.\n\nThe carbon framework of all polyketides is assembled by a polyketide synthase (PKS). Type I PKS modules act successively in polyketide chain elongation, processing, and termination. Each module contains acyl carrier protein (ACP), ketosynthase (KS), and acyltransferase (AT) domains that extend the linear sequence of an intermediate by two carbon atoms. The AT domain loads the ACP with a building block from a specific substrate, acyl-coenzyme A (acyl-CoA), and the KS domain catalyzes C–C bond formation between the intermediate from the upstream module and the acyl-ACP. In addition, modules may contain domains that have the ability to modify the β-keto group to a hydroxyl group, a double bond, or a single bond, in the presence of ketoreductase, dehydratase, and enoylreductase domains, respectively. Therefore, the modular organization of these enzyme assembly lines holds tremendous promise for applications in synthetic biology and bioengineering23,24. By engineering an appropriately modified PKS, new polyketides can be rationally designed and produced25. However, because the bacterial PKS module architecture and its conformational dynamics during polyketide chain elongation and processing are not yet well understood, the combinatorial biosynthesis of PKS often has been beset by many problems, such as inefficiency and dysfunction26.\n\nThus, for more than 10 years, enormous research efforts have focused on the structural biology of PKS, and consequently crystal structures have been reported for various excised PKS domains, KS-AT didomains, and docking domains27–37. In 2008, Maier et al. reported the crystal structure of porcine fatty acid synthase (FAS) and proposed a catalytic model of PKS38. Although modular PKSs are thought to share a common ancestor with mammalian FAS39, there are several differences between PKS and FAS. Recently, high-resolution information on the overall structure and organization of a complete PKS module was reported40,41. In this study, the crystal structure of a full-length PKS module from the pikromycin (Figure 2) pathway of Streptomyces venezuelae was determined by electron cryomicroscopy. The structural data revealed the dynamics of ACP during sequential interactions with catalytic domains within the reaction chamber, and when transferring the elongated and processed polyketide substrate to the next module in the PKS pathway. The ACP domain optimally localizes its cargo for the next reaction. This series of structures provided significant new information about the dynamics of ACP and its interaction with the catalytic domains, as well as a new reference for future analyses of substrate selectivity and catalytic efficiency42. The detailed insights of the PKS assembly line revealed in this study provide a new structural framework for the development of an effective approach for combinatorial biosynthesis.\n\nIn addition to PKS, non-ribosomal peptide synthetases (NRPSs) and PKS-NRPS hybrids represent a large multi-enzyme family that is involved in the biosynthesis of many microbial peptides and glycopeptide antibiotics, such as teicoplanin and vancomycin (Figure 2). Streptomyces spp. produce natural compounds, often with highly complex structures, which are difficult to produce or are not economically feasible to produce by chemical synthesis. Therefore, understanding biosynthetic pathways is of great clinical importance, and it is essential for combinatorial biosynthesis. Several months ago, Haslinger et al. reported the structure of a previously uncharacterized “X-domain”, which is strictly conserved in the final module of all glycopeptide NRPSs43. The X-domain interacts with two cytochrome P450 molecules, and the structures of the X-domain, both in isolation and in complex with the first P450 oxygenase protein involved in teicoplanin biosynthesis, revealed the inactive nature of the X-domain, as well as how oxygenase recruitment occurs.\n\nβ-lactam compounds account for more than half of the global antibiotic drug market44. However, the increased incidence of bacterial β-lactam resistance has led to an increasing reliance on a relatively new subclass of these drugs, which are known as carbapenems (Figure 2)44,45. Therefore, a better understanding of the biosynthetic mechanisms of carbapenems could facilitate further carbapenem-derived drug development to combat resistance. During the biosynthesis of these compounds, it is unclear whether carbapenem synthase (CarC) catalyzes the inversion of C5 in the β-lactam ring. By combining x-ray crystallography with multiple spectroscopic probes, Chang et al. recently revealed the mechanism by which the CarC enzyme inverts the precursor configuration of C5 to that of its mirror image46. A greater understanding of the structure and function of CarC is expected to aid in the efforts to bioengineer new carbapenems.\n\n\nThe secret of sulfur in natural products has been revealed\n\nMany natural products contain sulfur, which is a ubiquitous element that is essential for nutrition and metabolism in living systems. However, the mechanism by which this atom is incorporated into sulfur-containing compounds remains poorly understood.\n\nBE-7585A (Figure 2) is a 2-thiosugar-containing antibiotic that is produced by Amycolatopsis orientalis47. To study the production of the 2-thiosugar moiety in BE-7585A, Sasaki et al. identified a thiazole synthase homologue, BexX, which is responsible for the biosynthesis of 2-thiosugars in BE-7585A48. Briefly, as a 2-thioglucose synthase, BexX converts glucose-6-phosphate to 2-thioglucose. However, sulfur-carrier proteins, the donors of the sulfur moiety, are also involved in the biosynthesis of cysteine and molybdopterin, which are primary metabolites. In this case, sulfur transfer proteins from primary metabolic pathways are hijacked to facilitate the biosynthesis of secondary metabolites, which represents a rare link between primary and secondary metabolism.\n\nAnother study investigated the sulfur-containing compound lincomycin A (Figure 2), an efficient antibacterial that is produced by Streptomyces lincolnensis and used for agricultural purposes49. As is known, mycothiol (MSH) is a cysteinyl pseudo-disaccharide that is found in Gram-positive actinobacteria, and the conjugation of this thiol-containing compound to electrophilic toxins facilitates their excretion from the bacterial cell50. Ergothioneine (EGT), a histidine betaine derivative, is another thiol that is produced by actinomycetes51. In the biosynthesis of lincomycin A, both MSH and EGT play important roles in the incorporation of sulfur. EGT acts as a carrier in the template-guided molecular assembly, and MSH is the sulfur-donor for lincomycin maturation after thiol exchange52. In the biosynthesis of lincomycin A, these thiols function through two unusual S-glycosylations that program lincosamide transfer, activation, and modification, providing the first paradigm for EGT-associated biochemical processes and the poorly understood MSH-dependent biotransformations52. This study also demonstrated that the integration of primary metabolites (such as MSH and EGT) and secondary metabolites is crucial for the biosynthesis of complex molecules53.\n\n\nEtoposide: the next candidate for total biosynthesis in microbes?\n\nAs previously mentioned, numerous clinically used drugs, such as artemisinin and opioids, are derived from plant secondary metabolites. However, little is known about their biosynthetic genes, and this prevents highly efficient production of these compounds by engineered hosts. Since very few complete pathways of plant-derived drugs have been identified, heterologous production has been successfully applied for a few plant-derived drugs.\n\nMonths ago, the biosynthetic pathway of another efficient anti-cancer drug, etoposide (Figure 2), was identified54. As is known, etoposide is very difficult to obtain because its precursor is derived from the very slow-growing mayapple plant (Podophyllum peltatum), which is also an endangered species55. Therefore, Lau and Sattely worked out the pathway that produces the precursor of etoposide in the mayapple plant using bioinformatics, heterologous enzyme expression, and kinetic characterization54. Notably, only six enzymes are responsible for the complete biosynthesis of the etoposide aglycone by the mayapple plant. By transferring these six genes to the tobacco plant, the etoposide aglycone was successfully produced in a heterologous host.\n\n\nPerspectives\n\nBy reviewing the literature and some recent remarkable case studies of natural product biosyntheses or enzymatic functions, the significance and utility of enzymatic functions for synthetic biology applications can be demonstrated. As Charles Dickens said at the beginning of A Tale of Two Cities, “It was the best of times, it was the worst of times”, but now we have to admit that this is the best of times for synthetic biology. Thanks to the advent and widespread application of “-omics” techniques as well as computational biology techniques, more and more enzymes involved in natural product biosyntheses have been revealed. In addition, with the rapid emergence of new techniques, such as the latest powerful gene-editing tool CRISPR/Cas9, the roles of targeted genes that encode enzymes involved in natural product biosyntheses can also be efficiently identified. Finally, by using electron cryomicroscopy, structural biologists are now able to solve the complex catalytic mechanisms of enzymes. In an era of exponentially accumulating data, any optimization or innovation of a new technique could speed up the exploration of enzymatic functions. Consequently, more BioBricks or components are increasingly being deposited into the synthetic biology toolbox (Figure 3), which can be used in drug development and combinatorial biosynthesis by designing new routes, and merging biosynthetic and synthetic routes.", "appendix": "Competing interests\n\n\n\nThe authors declared no competing interests.\n\n\nGrant information\n\nThis work was funded by the National 973 Program of China (grants 2011CBA00800 and 2012CB721000), the National 863 Program of China (grant 2012AA02A701), the National Natural Science Foundation of China (grants 31170096, 312220170, and 31500072), the Natural Science Foundation of HuBei Province (grant 2015CFB415), and the China Postdoctoral Science Foundation (grant 2014M562052).\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nKinghorn AD, Pan L, Fletcher JN, et al.: The relevance of higher plants in lead compound discovery programs. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "11431", "date": "04 Dec 2015", "name": "Claudia Schmidt-Dannert", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "11432", "date": "04 Dec 2015", "name": "Gong-Li Tang", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/4-1399
https://f1000research.com/articles/5-216/v1	25 Feb 16	{ "type": "Research Article", "title": "Comparison of the effectiveness and side effects of dofetilide and dronedarone in the treatment of atrial fibrillation during an indicated period in time with perceived equipoise", "authors": [ "Nazem Akoum", "Feras Bader", "Maureen Ghanem", "David Stenehjem", "Zachary Burningham", "Brian C. Sauer", "Feras Bader", "Maureen Ghanem", "David Stenehjem", "Zachary Burningham", "Brian C. Sauer" ], "abstract": "Dronedarone is an anti-arrhythmic drug (AAD) originally approved for the treatment of atrial arrhythmias. The effectiveness and side effects of dronedarone have not been adequately compared to other commonly used AADs using observational data. We compared rates of recurrent atrial arrhythmias, incidence of side effects, and discontinuation rates of dronedarone to another class III AAD, dofetilide. We included patients from a single academic medical center between 2003 and 2010. Chart review was utilized to collect historical data of baseline clinical characteristics, side effects, arrhythmia recurrence, and drug discontinuation. Propensity score matching was used to balance baseline covariates. Cox-proportional hazard models were used to compare rates of recurrence between dronedarone and dofetilide. Patients were excluded if they failed to acutely achieve sinus rhythm, developed side effects leading to immediate discontinuation, or did not have sufficient follow-up. The final analysis included 127 dofetilide patients and 57 dronedarone patients. Fifty-nine patients (46.5%) experienced recurrence in the dofetilide group within the first year of treatment compared to 42 dronedarone patients (71.2%) (p<0.01). The adjusted hazard rate of recurrence was 2.42 times greater for dronedarone compared to dofetilide (95% CI: 1.44, 4.07; p-value<0.01). Side effects leading to drug discontinuation, including significant QT prolongation, developed more frequently with dofetilide (24.1% vs. 9.9%; p<0.01). Dronedarone is less effective than dofetilide in arrhythmia suppression. Our findings suggest dofetilide is associated with more serious side effects and a higher rate of discontinuation.", "keywords": [ "dofetilde", "dronedarone", "anti-arrhythmic", "atrial fibrillation", "arrhythmia", "side effects" ], "content": "Introduction\n\nA variety of anti-arrhythmic drugs (AADs) are used to suppress arrhythmias encountered in clinical practice1. AADs exert their pharmacologic effect by blocking ion channel currents in myocardial cells. Specifically, class III AADs block the delayed potassium rectifier current (IKr) and prolong phase 3 repolarization of the individual cell action potential. While this mechanism of action is aimed at suppressing arrhythmias, it can also predispose to other, potentially more dangerous ones. AADs also have important non-cardiac side effects. Despite being generally safe and well tolerated in the majority of patients, AADs demonstrate various degrees of efficacy in controlling arrhythmias in the clinical setting. The clinical use of AADs has been somewhat disappointing due to limited effectiveness and the occurrence of cardiac and systemic side effects.\n\nDofetilide is a class III AAD in clinical use for over ten years for the treatment of atrial arrhythmias. It is especially useful in patients with structural heart disease, such as coronary artery disease and congestive heart failure2. This is supported by clinical trials comparing its efficacy and safety to placebo3. Initiation of dofetilide requires hospitalization to ensure rigorous observation and monitoring for long QT, Torsade de Pointes, or other side effects through the first six doses. Patients are then followed up on a routine basis where they are monitored for drug efficacy and side effects.\n\nDronedarone is an additional class III AAD used for the treatment of atrial arrhythmias. It is a non-iodinated derivative of amiodarone, and it was originally believed to have a more favorable side effect profile compared to the parent drug. Dronedarone’s original approval was based on clinical trials demonstrating increased efficacy compared to placebo4,5. In the European and Australian-American-African trials EURIDIS and ADONIS respectively5, dronedarone significantly prolonged the time to first recurrence in patients with paroxysmal and persistent atrial fibrillation and flutter. However, one placebo-controlled clinical trial, referred to as the Permanent Atrial Fibrillation Outcome Study Using Dronedarone on Top of Standard Therapy (PALLAS), ended prematurely due to safety reasons, as the dronedarone arm showed significantly higher rates of major cardiovascular events6. A recent meta-analysis, comprised of seven placebo-controlled clinical trials, including the PALLAS trial, was implemented to better understand why the PALLAS trial’s findings conflicted with other similar investigations. This analysis reported significant heterogeneity of dronedarone treatment effects and concluded that permanent AF may be the most important predictor of harmful effects potentially caused by dronedarone7. Furthermore, a recent subgroup analysis was performed using the PALLAS trial, and investigators observed a harmful interaction between dronedarone and digoxin in patients with permanent AF, which they felt was partly responsible for the higher death rate observed in the original clinical trial8.\n\nCurrently, dronedarone is restricted or contraindicated in patients with symptomatic heart failure with New York Heart Association (NYHA) class II or III symptoms, recent heart failure hospitalization or left ventricular ejection fraction <40%, largely due to the results of the PALLAS trial9. However, dronedarone may still be an important treatment option for clinicians to consider when treating patients that do not meet these criteria and are subsequently experiencing paroxysmal or persistent AF. Strengthening the available evidence could potentially reduce the trial and error process clinicians maybe be following in prescribing AADs.\n\nTo our knowledge only two observational studies have been implemented to compare the efficacy and safety of dronedarone to other anti-arrhythmic drugs10,11. However, only one of these studies was able to directly compare dronedarone and dofetilide. This study suggested the efficacy of dronedarone and dofetilide to be similar, with dronedarone being associated with a great risk of cardiac-related admissions. This evidence could benefit from replication, as the study was restricted to a patient cohort generated from a single university medical center. Thus, further investigation is warranted in improving the generalizability of such findings, further characterizing dronedarone treatment effects in a ‘real world’ clinical setting. In addition, further investigation may also assist in helping us to better understand why the PALLAS trial observed such serious adverse drug events.\n\nTherefore, the goal of this study was to utilize a historical cohort to compare the effectiveness of dronedarone and dofetilide in maintaining normal rhythm. The results of the PALLAS trial were not known during the study period of our analysis and could not have influenced the recommendation or choice of anti-arrhythmic drugs. This study is comprised of a group of patients with comparable clinical characteristics in a large single center university hospital setting. We also compared the side effect profile and discontinuation rates of the two agents.\n\n\nMethods\n\nThe University of Utah institutional review board (IRB) approved this study (IRB#39735) and a waiver of informed consent was granted. We employed a historical cohort design using an intention-to-treat analysis designed to compare rates of arrhythmia recurrence and side-effects between dronedarone and dofetilide. Patients dispensed dronedarone or dofetilide were identified from the University of Utah’s hospital pharmacy database from January 2003 to 2010. Clinical pharmacists reviewed all patient charts using a structured template to extract relevant clinical variables including patient characteristics, clinical course, and response to drug therapy. Cohort entry was defined as the time of drug initiation. Patients initiated on either dronedarone or dofetilide that met inclusion criteria were considered on treatment for the drug they were initiated on until the end of follow-up or if they experienced an adverse event that required discontinuation. Inclusion criteria included initiation and long term follow up for drug monitoring at the University of Utah. The time window used to compare the outcomes of atrial arrhythmia recurrence, side effects, and discontinuation rates was restricted to the first year of drug treatment for both groups. Patients were excluded from the analysis if they did not receive their long-term follow-up care at the University of Utah. They were also excluded if they failed to achieve sinus rhythm or experienced significant adverse events leading to drug discontinuation within the first six doses of the medication.\n\nThe primary outcome was arrhythmia recurrence within the first year of treatment. Secondary outcomes included a comparison of side effects and drug discontinuation rates. Each patient’s experience on the drug was tracked through progress notes entered into the electronic medical records system. When arrhythmia recurrence was documented in progress notes, the patient was classified as having an event and censored from the recurrence analysis. Patients who did not achieve the primary outcome under drug exposure were censored at the end of the one-year follow-up period. Patients who developed a side effect from drug therapy but continued treatment without arrhythmia recurrence were not censored until the end of the one-year follow-up period. Patients who discontinued therapy due to adverse events were censored at the time of the adverse event.\n\nCovariates included in the analysis were selected based on patients’ clinical characteristics and comorbidities. These included patient age, gender, prior diagnoses of diabetes, hypertension, coronary artery disease, kidney function, congestive heart failure, concomitant drug treatment, and left ventricular ejection fraction. In addition, arrhythmia history and severity were included with the type of AF (paroxysmal or persistent), prior treatment with other AADs, or catheter ablation.\n\nDescriptive statistics were calculated and chi-square or t-tests for equality of means between treatment groups were computed before and after matching. One-to-one nearest neighbor propensity score matching was used to balance potential confounders between treatment groups. An analysis using propensity scores contains two steps: 1) Estimation of the probability of being treated (propensity score) using probit regression, and 2) Incorporation of the propensity score as a matching variable in the outcome model. All statistical analyses, including propensity scores and 1:1 nearest neighbor matching were computed using STATA 11 (STATA corp, College Station, Tx)12.\n\nPropensity scores are typically used to model the probability of being treated, and for this reason, they are used to calculate the average treatment effect in the treated. Since we are comparing two treatments, we modeled the probability of being treated with dronedarone – the newer agent being compared to the older therapy, dofetilide. In propensity score matching, it has been argued that inference should be restricted to areas where propensity scores overlap between treatment groups. For this reason, results are reported with and without imposition of common support (Figure 1). When the common support option is used, treatment observations (in this case dronedarone users) whose propensity score is higher than the maximum or less than the minimum propensity score of dofetilide users are dropped. Crude, covariate adjusted, and propensity score matched Cox-Proportional Hazard models were used to estimate hazard ratios for the difference in recurrence rates between treatments. Missing values for continuous measures were imputed using individual-level regression equations. Analyses were performed with and without imputed values and leaving the variables out of the equations all together. The hazard ratios; nevertheless, were not impacted in any meaningful way, and for this reason, we reported analyses with imputed values.\n\n\nResults\n\nDuring the period of January 2003 to September 2009, 162 patients were observed to have initiated dofetilide, while 71 patients were observed to have initiated dronedarone between September 2009 and September 2010. Direct comparison of baseline characteristics between the two drug groups described in Table 1 showed more females in the dronedarone group (40.9% vs. 25.9%, p=0.02). Dronedarone patients also had an older average age (68±12 vs. 62±13; p=0.001), a higher left ventricular ejection fraction (58±12 vs. 47±15; p<0.001), and were more likely to have undergone catheter ablation (53.5% vs. 24.1%; p<0.001). By contrast, a higher prevalence of congestive heart failure was seen in the dofetilide group (42.6% vs. 19.7%; p<0.001). An increased use of concomitant drug therapy was also observed in the dofetilide group with a higher use of beta-blockers, angiotensin-converting-enzyme inhibitors, aldosterone antagonists, and digoxin. The proportion of patients with paroxysmal atrial fibrillation was similar between the two groups (43.6% vs. 48.9%; p=0.48).\n\nPatients initiated on either drug had to achieve normal sinus rhythm in order to be maintained on long-term treatment and be included in the evaluation of recurrence. Sinus rhythm was present upon drug initiation in 70 dofetilide patients (43.2%) compared to 37 dronedarone patients (55.2%; p=0.09). Forty-five patients (27.8%) converted to sinus rhythm with dofetilide loading compared to 15 dronedarone patients (21.3%; p=0.32). Direct current cardioversion was used to achieve sinus rhythm, when drug loading failed to achieve this, in 19 dofetilide patients (11.7%) compared to 7 dronedarone patients (9.9%; p=0.67). In total, sinus rhythm was achieved in 134 dofetilide patients (82.7% of initial cohort) and 59 dronedarone patients (83.1% of initial cohort; p=0.94). In addition, if patients developed significant side effects upon drug initiation, the drugs were discontinued and not used for long-term arrhythmia suppression. Gastrointestinal side effects were significantly higher in the dronedarone group (11.3% vs. 1.9%; p<0.01) while QT prolongation was significantly increased in the dofetilide group (13% vs. 1.4%; p<0.001).\n\nAdequate follow-up information was available on 127 of 162 patients in the dofetilide group, and 59 of 71 patients treated in the dronedarone group. The average number of office visits during the first year of drug treatment was 4.5±1.2 for dofetilide patients compared to 4.2±1.7 for dronedarone patients (p=0.17). Fifty-nine patients (46.5%) experienced recurrence in the dofetilide group within the first year of treatment compared to 42 patients (71.2%) of dronedarone patients (Figure 2). A Kaplan Meier survival curve showing the difference in arrhythmia recurrence over the first year of treatment visually illustrates the difference in recurrence rates between dronedarone and dofetilide (Figure 3). Recurrence rates per 1000-days are reported in Table 2. A Cox proportional hazard model with the treatment drug assignment as the predictor variable was used to compare the two agents (Table 3). The hazard for one-year recurrence with dronedarone was 2.7 (95% CI: 1.79, 3.99; p-value<0.01) times larger than the hazard for dofetilide. The findings for the traditional covariate-adjusted model were similar, where the hazard ratio was 2.9 (95% CI: 1.74, 4.88; p-value<0.001).\n\niPS = Propensity Score\n\nkCS = Common Support\n\nPropensity score matching was also used to adjust for potential confounders. As seen in Supplementary material Table 1, significant differences existed in baseline characteristics between the 127 dofetilide and 59 dronedarone patients. The matching procedures were able to balance these covariates. This table also presents post-matching means and p-values when matches were restricted to regions of common support or not. The propensity score matched survival models produced similar hazard ratios to the crude and standard regression approaches. Shown in Table 3, the hazard for one-year recurrence in the dronedarone group was 2.9 (95% CI: 1.78, 4.84; p-value<0.00) times larger than the hazard for the dofetilide group when matching was not restricted to areas of common support and 2.4 (95% CI: 1.44, 4.07; p-value<0.00), when matching was restricted to areas of common support.\n\nDuring long-term follow-up, drug discontinuation due to concern with side effects was observed significantly more frequently in dofetilide patients compared to dronedarone patients (31 patients (24.4%) vs. 5 patients (8.5%); p<0.01). QT interval prolongation and ventricular arrhythmias (ventricular premature beats, sustained, and non-sustained ventricular tachycardia) were the most frequent causes of drug discontinuation in the dofetilide group (16 patients (10.2%), compared to 2 patients (3.4%) in the dronedarone group (p=0.11)). One patient in the dronedarone group demonstrated polymorphic ventricular tachycardia suggesting Torsade de Pointes. Gastrointestinal side effects were more frequent in the dronedarone group (6 patients (10.2%)) compared to the dofetilide group (2 patients (1.5%); p=0.03).\n\n\nDiscussion\n\nOur study compares the effectiveness and side effect profile of two class III anti- arrhythmic agents dronedarone and dofetilide. We demonstrate that dronedarone is associated with a significantly higher arrhythmia recurrence rate compared to dofetilide after one year of usage. However, we observed dofetilide to be associated with a significantly higher rate of serious side effects, specifically QT interval prolongation and ventricular arrhythmia.\n\nThe ideal way to compare the efficacy and tolerability of these two agents is a prospective randomized controlled trial, where all clinical covariates would be balanced through randomization and any patient would have an equal probability of being assigned to receive one of the two agents. The majority of current literature that has examined the efficacy and tolerability of dronedarone are placebo-controlled clinical trials. Due to the premature ending of the PALLAS trial, coupled with difficulties in the patient enrollment process surrounding cost and monitored care, an observational study design utilizing historical data is a reasonable and ethically appropriate alternative in comparing these agents. In addition, such a design can better reflect treatment effects that may occur in a ‘real world’ clinical setting, as clinical trials are performed in a highly controlled environment, often comprised of choice patient candidates.\n\nOur findings do not fully reflect the findings from the aforementioned University of Pittsburgh Medical Center (UPMC) observational investigation. This may be in part due to the fact that our measures of drug tolerability and safety differed. In addition, our sample sizes were not comparable and we did not examine multiple AADs, such as amiodarone and sotalol. Our lack of comparisons and subsequent smaller sample sizes are two notable limitations of our design. However, some may argue that UPMC design may be susceptible to chance statistical associations due to the multiple comparisons being made in their multivariate analyses. Furthermore, we attempted to handle statistical differences in baseline patient characteristics differently than the UPMC study: by means of the nearest neighbor one-to-one propensity score, which assisted us in reducing the effects of potential selection biases.\n\nOur study shows that dofetilide is more effective than dronedarone in preventing atrial arrhythmia recurrence in a ‘real world’ clinical setting. The probability of maintaining sinus rhythm with dofetilide at one year was 53.5% in our cohort. This is comparable to the reported probability of around 60% at 2 years in the DIAMOND-CHF trial, which successfully studied patients with congestive heart failure, in addition to their atrial fibrillation. Our rate of atrial fibrillation recurrence in the dronedarone group was comparable to that observed in the DIONYSOS trial [71.2% (n=71) vs. 63.5% (n=249); two sample test of proportions p=0.18]13. On the contrary, the University of Pittsburgh Medical Center (UPMC) retrospective cohort analysis reported dofetilide and dronedarone to have similar efficacy10. It is important to note that the DIONYSOS study findings are also in agreement with a portion of the findings from the UPMC study, suggesting amiodarone to be superior to dronedarone in preventing arrhythmia recurrence.\n\nWe also have demonstrated that dronedarone is better tolerated than dofetilide, which would appear to be contrary to what the PALLAS trial would suggest. This may in part be due to the multiple differences in baseline characteristics between patients in the PALLAS trial and those in our analysis, most notably in age, gender, and heart failure status. Moreover, unlike the PALLAS trial, none of our patients had permanent AF when drug therapy was initiated. Furthermore, despite the higher incidence of gastrointestinal side effects observed in our dronedarone group, this did not lead to higher rates of discontinuation. In our particular cohort, dronedarone was most likely continued because prescribing providers at the time were less concerned about life threatening side effects with this agent. Ventricular arrhythmias associated with long-term use of dofetilide were estimated at 10.2%, which is very similar to the rate observed in the DIAMOND-CHF trial (7.0%, including ventricular fibrillation, Torsade de Pointes, monomorphic, and polymorphic ventricular tachycardia). Side effects associated with dofetilide, specifically QT interval prolongation and subsequent pro-arrhythmia, led to a higher rate of drug discontinuation.\n\n\nConclusion\n\nThe findings in our study fill an important gap in the medical literature and demonstrate the comparative effectiveness of dronedarone to anti-arrhythmic agents other than amiodarone using observational data. Amiodarone is known as the most effective agent available, but other agents are often needed when long-term amiodarone use leads to subsequent side effects13,14. Furthermore, these findings suggest that dronedarone may actually be a well-tolerated treatment regimen for those with paroxysmal or persistent AF, after normal sinus rhythm has been achieved. However, clinicians should take into consideration patient-specific characteristics in order to reduce the risk of adverse events and complications. Despite the relatively small sample size of patients included in our study, our results are robust and provide important clinical evidence that will aid providers in managing patients with atrial fibrillation. Randomized prospective trials designed to compare the two agents may be difficult to conduct at this time and further observational studies, possibly from multiple centers, will continue to strengthen the evidence generated from this study.\n\n\nData availability\n\nRaw datasets are not available due to the regulations surrounding patient data; University of Utah IRB approval and agreement to all related data use policies is required. This data can be obtained by contacting the authors, University of Utah IRB, and Department of Internal Medicine, with the University of Utah. Instructions for applying for IRB approval are available on the following University of Utah ERICA website (http://irb.utah.edu/guidelines/erica-assistance/access-instructions.php). An individual must first obtain a University of Utah ID (uNID) number as outlined and instructed on the ERICA website. The Department of Internal Medicine, within the University of Utah, will then approve the request for issuing an individual with a uNID. Once a uNID is obtained, the individual can then create a UU IRB ERICA account. Upon completion of self-registering in ERICA, the individual can be granted access to the study and de-identified data via the University of Utah IRB number that can be obtained from the authors.", "appendix": "Author contributions\n\n\n\nNA conceived the study, carried out the research, and prepared initial drafts of the manuscript. FB assisted in designing the study, provided critical review, and participated in editing the manuscript. MG and DS assisted with the data collection, provided critical review, and participated in editing the manuscript. ZB polished initial manuscript drafts, added critical epidemiologic review, and prepared the manuscript for submission. BS provided oversight and critical review of the study design prior to implementation, assisted in carrying out the research and analysis, and participated in drafting sections of the manuscript relevant to his expertise.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nSupplementary material\n\nThe distributions have been summarized with and without restricting matching to areas of common support.\n\n\nReferences\n\nFuster V, Rydén LE, Cannom DS, et al.: 2011 ACCF/AHA/HRS focused updates incorporated into the ACC/AHA/ESC 2006 guidelines for the management of patients with atrial fibrillation: a report of the American College of Cardiology Foundation/American Heart Association Task Force on practice guidelines. Circulation. 2011; 123(10): e269–367. PubMed Abstract \| Publisher Full Text\n\nKøber L, Bloch Thomsen PE, Møller M, et al.: Effect of dofetilide in patients with recent myocardial infarction and left-ventricular dysfunction: a randomised trial. Lancet. 2000; 356(9247): 2052–8. PubMed Abstract \| Publisher Full Text\n\nPedersen OD, Bagger H, Keller N, et al.: Efficacy of dofetilide in the treatment of atrial fibrillation-flutter in patients with reduced left ventricular function: a Danish investigations of arrhythmia and mortality on dofetilide (diamond) substudy. Circulation. 2001; 104(3): 292–6. PubMed Abstract \| Publisher Full Text\n\nHohnloser SH, Crijns HJ, van Eickels M, et al.: Effect of dronedarone on cardiovascular events in atrial fibrillation. N Engl J Med. 2009; 360(7): 668–78. PubMed Abstract \| Publisher Full Text\n\nSingh BN, Connolly SJ, Crijns HJ, et al.: Dronedarone for maintenance of sinus rhythm in atrial fibrillation or flutter. N Engl J Med. 2007; 357(10): 987–99. PubMed Abstract \| Publisher Full Text\n\nConnolly SJ, Camm AJ, Halperin JL, et al.: Dronedarone in high-risk permanent atrial fibrillation. N Engl J Med. 2011; 365(24): 2268–76. PubMed Abstract \| Publisher Full Text\n\nHohnloser SH, Connolly SJ, John Camm A, et al.: An individual patient-based meta-analysis of the effects of dronedarone in patients with atrial fibrillation. Europace. 2014; 16(8): 1117–24. PubMed Abstract \| Publisher Full Text\n\nHohnloser SH, Halperin JL, Camm AJ, et al.: Interaction between digoxin and dronedarone in the PALLAS trial. Circ Arrhythm Electrophysiol. 2014; 7(6): 1019–25. PubMed Abstract \| Publisher Full Text\n\nJanuary CT, Wann LS, Alpert JS, et al.: 2014 AHA/ACC/HRS guideline for the management of patients with atrial fibrillation: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines and the Heart Rhythm Society. J Am Coll Cardiol. 2014; 64(21): e1–76. PubMed Abstract \| Publisher Full Text\n\nQin D, Leef G, Alam MB, et al.: Comparative effectiveness of antiarrhythmic drugs for rhythm control of atrial fibrillation. J Cardiol. 2015; pii: S0914-5087(15)00207-5. PubMed Abstract \| Publisher Full Text\n\nAllen LaPointe NM, Dai D, Thomas L, et al.: Antiarrhythmic drug use in patients <65 years with atrial fibrillation and without structural heart disease. Am J Cardiol. 2015; 115(3): 316–22. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLeuven E, Sianesi B: Psmatch2: STATA module to perform full Mahalanobis and propensity score matching, common support graphing, and covariate imbalance testing. 2003. Reference Source\n\nLe Heuzey JY, De Ferrari GM, Radzik D, et al.: A short-term, randomized, double-blind, parallel-group study to evaluate the efficacy and safety of dronedarone versus amiodarone in patients with persistent atrial fibrillation: the DIONYSOS study. J Cardiovasc Electrophysiol. 2010; 21(6): 597–605. PubMed Abstract \| Publisher Full Text\n\nSingh BN, Singh SN, Reda DJ, et al.: Amiodarone versus sotalol for atrial fibrillation. N Engl J Med. 2005; 352(18): 1861–72. PubMed Abstract \| Publisher Full Text" }	[ { "id": "14713", "date": "13 Jul 2016", "name": "Richard Wyss", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 implements a historical cohort design to compare the effectiveness of dronedarone vs dofetilide with respect to arrhythmia recurrence and side effects within the first year of treatment using the University of Utah’s hospital pharmacy database. This study addresses an important topic as there is little empirical evidence comparing the real world effectiveness of dofetilide vs dronedarone. I believe that the paper would benefit, however, if a few methodological aspects of the study were described in greater detail:\nThe authors mention that follow-up started at the time of drug-initiation (dofetilide or dronedarone). It would be helpful if the authors explicitly mentioned if this was the first initiation of these specific drugs for each subject, or if there was a washout period prior to the initiation of either dofetilide or dronedarone where subjects were required to have no pharmacy claim of either drug (new-user design)?\n\nIt would be helpful if the authors explicitly defined in the Methods section what side effects and adverse events are being considered.\n\nThe authors mention that they performed PS matched analyses with and without imposition of common support (i.e., trimming of the non-overlapping regions of the PS distributions). It is unclear why this is necessary. 1-1 PS matching should automatically restrict the analysis to individuals where there is common overlap as individuals with no common support would not be matched. It would be helpful if the authors explained why PS matched analyses with and without the common support region defined prior to matching would be expected to change the matched population and give different answers.\n\nThe authors do a nice job of highlighting the small sample size of the study as a limitation in the analysis. I think that it would be important for the authors to also highlight the potential for unmeasured confounding in this study as well as residual confounding caused by the measured baseline covariates even after PS adjustment.\n\nTable 1 and Figure 1 show strong differences in baseline covariates and strong separation in PS distributions across treatment groups. This often implies strong differences in unmeasured factors as well. The potential for unmeasured confounding should at least be mentioned as a limitation in the Discussion.\n\nEven after PS adjustment, Supplemental Table 1 shows that there are still some strong differences in baseline covariates. None of these differences are reported as significant according to the P-value, but the P-value is highly affected by sample size and is not the best metric for assessing whether or not balance is achieved. When just looking at absolute mean differences in the baseline covariates, some of the differences do not improve after matching (e.g., Diabetes, CAD, HTN). Consequently, I think the potential for residual confounding should be mentioned as a limitation.", "responses": [ { "c_id": "2130", "date": "05 Aug 2016", "name": "Brian Sauer", "role": "Author Response", "response": "We want to thank Dr. Richard Wyss for his outstanding review our our manuscript and we would like to clarify some of the points and issues he raised. Unfortunately, this was not an inception cohort and patients may have had a previous exposure at the time they were indexed into our study. We will clarify this in the manuscript. It is possible that patients indexed on Dronedarone may have had a previous AAD. If they had a previous exposure to an AAD and failed they may be more likely to fail on dronedarone, which would produce some selection bias. That said, we don't believe the lack of effectiveness is driven by this form of selection bias because, at the time, the cardiology program essentially switched practice to use dronedarone whenever indicated. We will review and revise the methods section on ADEs abstracted from clinical notes. Because the PS matching was based on nearest neighbor and not a caliper distance it is possible to match a dofetilide subject to a dronedarone patient who is not an ideal counterfactual substitute. We currently prefer the use of caliper distance or matched weights but at the time we conducted this study we were using a nearest neighbor matching algorithm. The findings are very robust and the matching algorithm is not expected to qualitatively change the results of this study. We do agree that unmeasured confounding and residual confounding are of concern. It is important to understand that the prescribing practices of this institution changed from using dofetilide to dronedarone for indicated patients. This means providers for the most part were prescribing based on preference instead of patient risk. As a result, there could be historical bias where diagnostic or other care processes changed over time. We had much discussion on this issue and didn't believe there were substantial historical forms of bias, but this cannot be ruled out. Dr Wyss rightfully is concerned with residual confounding, especially because some of the covariate may have clinically meaningful unbalance between groups. I don't believe any of the post-matching (common support) comparisons remained statistically significant. Nevertheless, we should have presented the standardize difference score instead of the p-value, which is not the best way of evaluating balance especially with the smaller sample size. Even though residual confounding is likely the findings were consistent with clinical experience and remained stable across various statistical treatment." } ] }, { "id": "15544", "date": "30 Aug 2016", "name": "Mohammad Shenasa", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study is well designed and written during a long period of 7 years from 2003-2010. The study is a retrospective chart review and not a randomized trial as acknowledged by the authors.\nIt is not clear how follow-up and recurrence rates were identified (i.e. Holter monitoring, phone calls to patients, etc.).\n\nDid any patients in each group have an implantable rhythm management device (i.e. pacemaker or implantable cardioverter-defibrillator)? Were there any crossovers from dofetilide to dronedarone or vice versa?\n\nTable one shows almost 20% (19.7%) of patients with congenital heart failure were on dronedarone. The PALLAS trial demonstrated that the drug increased mortality; therefore, dronedarone should not be prescribed to such patients.\n\nThe authors should mention and further discuss that this data implied that dofetilide is more effective than dronedarone is in preventing recurrence of atrial arrhythmia.", "responses": [ { "c_id": "2191", "date": "19 Sep 2016", "name": "Nazem Akoum", "role": "Author Response", "response": "We thank Dr Mohammad Shenasa for reviewing our work and providing valuable commentary. We would like to answer the question he raised. Follow up was through outpatient clinic visits every 3 months and additional visit as clinically indicated based on patient symptoms. The average number of visits during the first year of treatment was 4.5±1.2 for dofetilide patients and 4.2±1.7 for dronedarone patients. 12 patients in the dofetilide group (7.4%) and 5 patients in the dronedarone (7.0%) had implantable rhythm management devices (p=0.91). This did not influence arrhythmia ascertainment for recurrence as arrhythmia episodes were detected at the scheduled clinic visits as described in point number 1 above. Remote rhythm monitoring through implantable rhythm devices was not available during the study period. Patients were censored from the recurrence analysis at the time of arrhythmia recurrence and therefore cross over, if it occurred, did not affect the study endpoints. As mentioned in the discussion section, the time period analyzed preceded the publication of the PALLAS trial, so excluding patients with heart failure from dronedarone use was not the standard of care at the time period analyzed. Our study shows that dofetilide is more effective than dronedarone in preventing recurrent atrial arrhythmia and this is clearly mentioned in the discussion. We acknowledge that prospective randomized studies are needed to further strengthen the evidence for this outcome." } ] } ]	1	https://f1000research.com/articles/5-216
https://f1000research.com/articles/4-1103/v1	23 Oct 15	{ "type": "Research Article", "title": "Adrenocorticotropic hormone gel in the treatment of systemic lupus erythematosus: A retrospective study of patients.", "authors": [ "Xiao Li", "Josh Golubovsky", "Joyce Hui-Yuen", "Ummara Shah", "Ewa Olech", "Rosalia Lomeo", "Vijay Singh", "Howard Busch", "Mary Jane Strandberg", "Kayla Strandberg", "Leslie Horowitz", "Anca Askanase", "Xiao Li", "Josh Golubovsky", "Joyce Hui-Yuen", "Ummara Shah", "Ewa Olech", "Rosalia Lomeo", "Vijay Singh", "Howard Busch", "Mary Jane Strandberg", "Kayla Strandberg", "Leslie Horowitz" ], "abstract": "Objectives: Acthar Gel is a long-acting formulation of adrenocorticotropic hormone (ACTH) with anti-inflammatory effects thought to be mediated in part through melanocortin receptor activation. This study was initiated to understand the role of Acthar Gel in SLE treatment in rheumatology practices.Methods: This is a retrospective case series of nine adult female patients treated with Acthar Gel for at least six months at five academic centers. Treating physicians completed a one-page questionnaire on lupus medications, disease activity, and outcomes. Clinical response was defined using SLEDAI 2K and improvement in the clinical manifestation(s) being treated.Results: The most common clinical SLE manifestations/indications requiring therapy with Acthar Gel were arthritis, rash, and inability to taper corticosteroids. The mean SLEDAI 2K score at baseline was 5.8 (range 0-16). Six patients were concomitantly treated with corticosteroids (mean dose 18.3mg/day). All patients were on background SLE medications including immunosuppressives. Seven of nine patients had an overall improvement, with a decrease in SLEDAI 2K to 3.5; four of five patients had improvement or resolution in arthritis, and one of two patients had resolution of inflammatory rash. Four patients discontinued corticosteroids and one patient tapered below 50% of the initial dose by 3 months of treatment with Acthar Gel. No adverse events were reported.Conclusions: This study suggests a role for Acthar Gel as an alternative to corticosteroids in the treatment of SLE. Acthar Gel appears to be safe and well-tolerated after 6 months of treatment, with a significant reduction in disease activity.", "keywords": [ "ACTH", "Acthar Gel", "lupus treatment", "SLE", "steroid-sparing agent", "Systemic Lupus Erythematosus" ], "content": "Introduction\n\nSystemic lupus erythematosus (SLE) is a chronic autoimmune disease of unknown etiology. The prevalence of SLE in the U.S. is estimated to be as high as 73 per 100,000 people1. The hallmark of SLE is the production of autoantibodies directed against the patient’s own healthy tissue and organs. Clinically, SLE is characterized by flares, periods of increased disease activity and periods of quiescence2.\n\nAdrenocorticotropic hormone (HP Acthar® Gel, repository corticotropin injection, Questcor Pharmaceuticals, Inc., Union City, CA) is FDA-approved for the treatment of SLE3. Acthar Gel was widely used in the 1950s as an effective therapy for SLE. However, it has been gradually replaced by synthetic glucocorticoid analogues. Acthar Gel is a long-acting formulation of adrenocorticotropic hormone (ACTH). ACTH is a 39 amino acid peptide that derives from post-translational processing of the precursor molecule proopiomelanocortin (POMC) and belongs to an anti-inflammatory group called melanocortins. Current understanding of Acthar Gel indicates that its effectiveness is a result of both its steroidogenic and direct anti-inflammatory effects through activation of different melanocortin receptors (MCRs)4–6. Acthar Gel binds to melanocortin receptors in a variety of cell types including immune cells. MC2R activation is responsible for the steroidogenic effects of Acthar Gel. Endogenous ACTH and Acthar Gel stimulate the adrenal cortex to secrete cortisol and other steroids by binding to the MC2R4. The trophic effects of endogenous ACTH on the adrenal cortex are not well understood beyond the fact that they appear to be mediated by cyclic adenosine monophosphate (cAMP). The release of endogenous ACTH is under the influence of the nervous system via the regulatory hormone released from the hypothalamus and by a negative corticosteroid feedback mechanism. Elevated plasma cortisol suppresses pituitary ACTH release. Administration of exogenous ACTH via Acthar Gel can over-ride the negative feedback mechanism4. There is a significant body of evidence as to which melanocortin receptor subtypes are involved in the direct anti-inflammatory effects7. MCRs are expressed on virtually all the cells; the activations of MC1R, MC3R, and MC5R, in particular, are thought to be responsible for the direct anti-inflammatory effect. This is supported by experiments using MCR-selective synthetic analogs, and animal data4,7–11. The MC1R-selective agonists inhibited tumor necrosis factor α (TNF-α)-induced activation of NF-κB and down-regulated expression and secretion of endothelial cell selectin, vascular cell adhesion molecule, and intercellular adhesion molecule in human dermal vascular endothelial cells treated with TNF-α4,11. In a study with adrenalectomized rats, Acthar Gel decreased experimental arthritis, indicating a steroid-independent action12.\n\nA recently published open-label trial by Fiechtner and Montroy evaluated ten SLE female patients with persistent moderate-to-severe active disease while receiving standard therapy treated with Acthar Gel, 1 mL (80 IU/mL) by self-administered subcutaneous injection for 7–15 days. These patients showed significant improvement in the intensity of flares (primary endpoint), as measured by the SLE Disease Activity Index (SLEDAI 2K) score13. Other clinical parameters also showed significant (p<0.05) improvement, including physicians’ and patients’ global assessments, fatigue score measured by the Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-Fatigue) scale, and erythrocyte sedimentation rate13.\n\nSLE standard of care treatment uniformly includes corticosteroids. However, the potential for serious side effects from long-term corticosteroid use motivates the development of clinical trials with steroid-free regimens. The desired immune suppression of steroids can increase the risk of infections and metabolic effects such as diabetes, significant weight gain, Cushingoid habitus (moon face, buffalo hump, truncal obesity), striae, increased blood pressure, and fluid retention are concerning to both patients and doctors. Long-term steroid use can also lead to other serious consequences such as osteoporosis and fractures, avascular necrosis, and cataracts. Acthar Gel stimulates the production of endogenous steroids, the overproduction of which would have similar side effects as the synthetic steroids used in the current SOC regime. However, the combination of steroid-mediated immunosuppressive and direct anti-inflammatory effects of Acthar Gel provides a different approach in the management of SLE activity. This study was initiated to further understand the role of Acthar Gel in the treatment of SLE in several rheumatology practices.\n\n\nMethods\n\nThis is a retrospective case series of patients from five academic/community clinical practices located in the United States. Five physicians from practices in Florida, Nevada, New York, and Virginia, agreed to participate in the study and complete a one-page questionnaire regarding the use of Acthar Gel in their patients with SLE. The study was approved by the Columbia University Medical Center Institutional Review Board and requisition of informed consent was waived.\n\nPatients in this study had been diagnosed with SLE by meeting at least four of eleven American College of Rheumatology (ACR) criteria or the Systemic Lupus International Collaborating Clinics (SLICC) classification criteria14,15. Based on the SLICC criteria, a patient is classified with SLE if four of the clinical and immunologic criteria are satisfied, including at least one clinical criterion and one immunologic criterion, or the patient has biopsy-proven nephritis compatible with SLE and with ANA or anti-dsDNA antibodies15. All patients were between 18 and 88 years of age, had taken or were currently taking Acthar Gel to treat SLE and were followed by their physicians for at least 6 months since initiation of Acthar Gel treatment. All patients had been on stable doses of immunosuppressants, including five patients on oral corticosteroids, for at least four weeks prior to initiation of Acthar Gel therapy. All patients had failed to respond clinically to multiple immunosuppressants and/or were taking immunosuppressants at the time of Acthar Gel initiation. Patients who were not on stable doses of immunosuppressants, including corticosteroids, for at least four weeks prior to initiation of Acthar Gel were excluded.\n\nThe questionnaire was a one-page survey that included information on demographics, SLE (treatment, disease activity, and laboratory results) and the administration of Acthar Gel (dose, duration of treatment, and side effects/adverse events). These data were recorded over six months of retrospective follow-up by the investigators, where the baseline questionnaire was recorded at time of initiation of Acthar by the treating physician. Physicians were asked to provide information on the clinical manifestations of patients being treated with Acthar Gel and whether those manifestations had improved at three and six months after initiation of therapy.\n\nSystemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI 2K) was calculated by the treating physician for each patient at baseline, 3 months, and 6 months after initiation of therapy with Acthar Gel. Improvement or resolution in the clinical manifestation(s) being treated was defined as follows: Improvement in arthritis was defined as a decrease by ≥50% in the number of swollen and tender joints and resolution was defined as the absence of swollen and tender joints. Improvement in rash was defined as a decrease in inflammatory rash by ≥50% of the affected area and resolution was defined as the absence of rash. These were in line with the SLEDAI 2K responder index (SRI-50), a modification of the SLEDAI that attempts to capture 50% improvement16. Per Aspreva clinical trial guidelines, partial response for lupus nephritis was defined as ≥50% improvement in proteinuria and complete response as proteinuria lower than 500mg/24 hours17. Improvement in all other initial symptoms driving the initiation of Acthar Gel was defined as ≥50% improvement per treating physician’s assessment. The definition of clinical response also included a decrease in ≥50% of the initial corticosteroid daily dose and/or discontinuation of corticosteroids during the time of treatment with Acthar Gel. Responders were the patients who achieved the outcome measures as defined above; non-responders were those who did not. Moreover, to be classified as a responder, there could be no worsening in other organ systems.\n\nData were collected on demographic and disease characteristics, clinical manifestations requiring treatment, concomitant medications, disease course, and treatment outcomes. Due to small sample size, descriptive statistics were used where appropriate (Graphpad Prism 6, La Jolla, CA). Indications for the initiation of Acthar Gel treatment, mean values of dose, and frequency of Acthar Gel treatment were evaluated. Data on response to Acthar Gel treatment and adverse events were summarized.\n\n\nResults\n\nTable 1 summarizes patient demographic data, disease history, and SLE clinical manifestations along with concomitant medications of the nine patients included in the study. Arthritis, inability to taper corticosteroids, and rash appeared to be the most common reasons for the initiation of Acthar Gel treatment. All nine patients had positive antinuclear antibodies (ANA) titers and the mean dose of 80 IU of Acthar Gel biweekly by subcutaneous injections was prescribed per recommended dosing18. Acthar Gel was added in addition to the patients’ immunosuppressant and steroid regimens to better control disease activity and to allow for prednisone taper.\n\naF-Female, bCNS-Central Nervous System, cNA-Not Available.\n\nEight of nine patients had follow-up data available through six months of follow-up. Of these patients, five had arthritis, five were taking prednisone, two had rash, two had pleurisy, and two had active nephritis at baseline (Table 1). The average corticosteroid dose at baseline was 18.3mg/day (range 7.5 to 40mg/day) and the average baseline SLEDAI 2K score was 5.8 (range 0 to 16). Patients with SLEDAI 2K scores of 0 were treated with Acthar Gel for inability to taper steroid, which was defined as requirement for prednisone treatment with >7.5mg for longer than 3 months despite repeated attempts at dose reduction. All other patients had persistent active disease.\n\nThere was an overall decrease by 40.2% in individual SLEDAI 2K scores in eight patients at Month 6. Two patients showed improvement in arthritis after six months of treatment, and two showed resolution of arthritis (Table 1). One of the two patients with renal involvement at baseline achieved complete remission of lupus nephritis at 3 months but relapsed with return of proteinuria and active lupus nephritis at 6 months. Of the five patients who were initially on prednisone, four were able to completely stop prednisone at 3 months after the initiation of Acthar Gel and remained off corticosteroids at 6 months. The one patient who remained on prednisone was able to taper prednisone from 30mg/day to 6mg/day in three months and then 2mg/day after six months without worsening arthritis. One patient had only 3-month follow-up data available due to being lost to follow-up; for this patient, corticosteroids were discontinued at 3 months after initiation of Acthar Gel therapy.\n\nOverall, seven of the nine patients in this study improved based on the ability to taper steroids, decrease in SLEDAI2K score, and the extent of their active clinical manifestations that had not resolved. Common side effects seen with use of Acthar Gel in other diseases include hypertension, hyperglycemia, increased susceptibility to infection, weight gain, and decreased bone density. However, no adverse effects were reported by any of the patients in our study while on treatment with Acthar Gel.\n\n\nDiscussion\n\nThe current study evaluates the efficacy and safety of Acthar Gel in nine patients with SLE from five academic clinical practices. Treatment with Acthar Gel improved disease activity and allowed patients to taper and discontinue steroids without significant side effects. In recent years, there has been increased interest in treatment regimens that allow for lower corticosteroids doses in SLE due to concerns about the serious long-term consequences of chronic corticosteroid therapy which are responsible for some of the damage accumulation in SLE. While part of Acthar Gel’s efficacy is mediated through the production of steroids from the adrenal cortex, the non-steroidogenic anti-inflammatory effects via MCR activation are the most interesting. Studies in animal models of inflammation suggest responses to ACTH in the absence of adrenal steroid production12. Additionally, Bomback et al.18 have demonstrated previously that cortisol levels after administration of Acthar Gel were within normal limits, supporting a limited contribution for cortisol in responses to Acthar Gel, at least in this population of patients on long-term prednisone therapy. Plasma cortisol levels were not evaluated in our study.\n\nA recently published open-label trial by Fiechtner and Montroy showed that Acthar Gel provided a significant reduction in SLE disease activity in ten patients13. Despite the small sample sizes, patients showed improvement across all disease manifestations. Our data further substantiate the efficacy of Acthar Gel in patients with a variety of SLE manifestations and a role for ACTH as a steroid-sparing agent. Further studies in a larger population and for longer study duration will be of benefit to examine long-term outcomes and delineate adverse events.\n\nAlthough data are limited, Acthar Gel appears to be well-tolerated, as no serious or unexpected adverse events were observed in our study patients, and the results of this study were consistent with historical observations and data from Fiechtner et al.13. Fiechtner et al.13 reported a sinus infection in one patient during the trial that resolved with antibiotic treatment, and another patient had bilateral edema in the lower extremities that resolved two weeks after the end of treatment.\n\nWe readily acknowledge several limitations of the study: the retrospective data collection, the small number of patients, the limited follow-up duration, and the lack of data on serum cortisol levels.\n\nIn conclusion, the results of this case series suggest that Acthar Gel is an effective therapeutic option for patients with SLE. Despite the limited number of patients, this study offers consistent results that afford an intriguing insight into the effectiveness of Acthar Gel in SLE and suggest a role for ACTH in the treatment of lupus as an alternative to oral corticosteroids. Acthar Gel is currently being investigated in clinical trials enrolling patients with persistently active SLE, proliferative lupus nephritis (Class III, IV) administered together with mycophenolate mofetil, and in patients with membranous lupus nephritis (Class V). The results of these randomized, controlled trials are eagerly awaited (NCT01753401, NCT02226341 and NCT01926054).", "appendix": "Author contributions\n\n\n\nAA, US, EO, RL, VS, and HB were involved in study conception and design. AA, US, EO, RL, VS, HB, MJS, KS, and LH were involved in patient enrollment and data collection. XL, JG, and AA analyzed the data. XL, JH, and AA contributed equally to the writing of this manuscript and data verification. All authors were involved in revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nAA is a consultant for Questcor Pharmaceuticals, Inc. XL, JG, JH, US, EO, RL, VS, HB, MJS, KS and LH declare no conflicts of interest.\n\n\nGrant information\n\nThe authors declared that no funding was involved in supporting this work.\n\n\nReferences\n\nLim SS, Drenkard C, McCune WJ, et al.: Population-based lupus registries: advancing our epidemiologic understanding. Arthritis Rheum. 2009; 61(10): 1462–6. PubMed Abstract \| Publisher Full Text\n\nBertsias G, Cervera R, Boumpas DT: Systemic lupus erythematosus: pathogenesis and clinical features. In: Bijlsma JWJ ed. EULAR Textbook on Rheumatic Diseases. London UK: BMJ Group, 2014; 476–505. Reference Source\n\nQuestcor Pharmaceuticals. H.P. Acthar® Gel (repository corticotropin injection). [prescribing information] Hayward, CA: Questcor Pharmaceuticals, Inc.; [updated September, 2012]. Reference Source\n\nCatania A, Gatti S, Colombo G, et al.: Targeting melanocortin receptors as a novel strategy to control inflammation. Pharmacol Rev. 2004; 56(1): 1–29. PubMed Abstract \| Publisher Full Text\n\nSi J, Ge Y, Zhuang S, et al.: Adrenocorticotropic hormone ameliorates acute kidney injury by steroidogenic-dependent and -independent mechanisms. Kidney Int. 2013; 83(4): 635–46. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGetting SJ: Targeting melanocortin receptors as potential novel therapeutics. Pharmacol Ther. 2006; 111(1): 1–15. PubMed Abstract \| Publisher Full Text\n\nMontero-Melendez T, Madeira MFM, Norling LV, et al.: Association between periodontal disease and inflammatory arthritis reveals modulatory functions by melanocortin receptor type 3. Am J Pathol. 2014; 184(8): 2333–41. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPatel HB, Bombardieri M, Sampaio AL, et al.: Anti-inflammatory and antiosteoclastogenesis properties of endogenous melanocortin receptor type 3 in experimental arthritis. FASEB J. 2010; 24(12): 4835–43. PubMed Abstract \| Publisher Full Text\n\nTaylor AW, Kitaichi N, Biros D: Melanocortin 5 receptor and ocular immunity. Cell Mol Biol (Noisy-le-grand). 2006; 52(2): 53–9. PubMed Abstract\n\nTaylor AW, Lee D: Applications of the role of α-MSH in ocular immune privilege. Adv Exp Med Biol. 2010; 681: 143–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGong R: The renaissance of corticotropin therapy in proteinuric nephropathies. Nat Rev Nephrol. 2011; 8(2): 122–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGubner R, Cote L, Hughes J, et al.: Comparative effects of aminopterin, cortisone and ACTH in experimental formaldehyde arthritis and psoriatic arthritis. J Invest Dermatol. 1952; 19(4): 297–305. PubMed Abstract \| Publisher Full Text\n\nFiechtner JJ, Montroy T: Treatment of moderately to severely active systemic lupus erythematosus with adrenocorticotropic hormone: a single-site, open-label trial. Lupus. 2014; 23(9): 905–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHochberg MC: Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 1997; 40(9): 1725. PubMed Abstract \| Publisher Full Text\n\nPetri M, Orbai AM, Alarcón GS, et al.: Derivation and validation of the Systemic Lupus International Collaborating Clinics classification criteria for systemic lupus erythematosus. Arthritis Rheum. 2012; 64(8): 2677–86. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTouma Z, Gladman DD, Ibañez D, et al.: Systemic Lupus Erythematosus Disease Activity Index 2000 Responder Index-50 enhances the ability of SLE Responder Index to identify responders in clinical trials. J Rheumatol. 2011; 38(11): 2395–9. PubMed Abstract \| Publisher Full Text\n\nAppel GB, Contreras G, Dooley MA, et al.: Mycophenolate mofetil versus cyclophosphamide for induction treatment of lupus nephritis. J Am Soc Nephrol. 2009; 20(5): 1103–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBomback AS, Canetta PA, Beck LH Jr, et al.: Treatment of resistant glomerular diseases with adrenocorticotropic hormone gel: a prospective trial. Am J Nephrol. 2012; 36(1): 58–67. PubMed Abstract \| Publisher Full Text" }	[ { "id": "10908", "date": "01 Feb 2016", "name": "Giovanni Franchin", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nXiao Li et al present a well written report on a retrospective series of nine patient with active lupus and the effect of ACTH gel. Although the study is limited by the small number of subjects they include a diverse patient population the majority of which had very active disease with an average baseline SLE Disease Activity Index 2K of 6 and on combination immunosuppressive therapy. Moreover, the patients represent our usually more challenging patients with inability to taper steroids and multiple organ involvement. The authors were careful to select patients who had been initiated in ACTH gel and had not had any recent adjustment of immunosuppressive therapy, which is a common confounder of disease response/activity. The results in terms of tapering down/off steroids is compatible with our understanding of the steroidogenic effect of Acthar gel. The improvement of SLEDAI 2K was seen for half of patients. It would have been reassuring if data on individual dosing of Acthar gel had been given for all patients and confirmation of drug administration.Overall this study offers strong observational data to support further studies into the effectiveness of ACTH analogues in the treatment of lupus.", "responses": [ { "c_id": "1799", "date": "05 Feb 2016", "name": "Joyce Hui-Yuen", "role": "Author Response", "response": "Thank you very much for your review. We appreciate your time in reviewing our article and your critical comments. We apologize for any confusion regarding the dose of Acthar, which was, in fact, 80 IU subcutaneously twice weekly in all patients. Unfortunately, administration of the drug was not confirmed in this study.Thank you again - we very much appreciate your time and effort in reviewing our article." } ] }, { "id": "11900", "date": "19 Feb 2016", "name": "Anne Eberhard", "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 9 patients who were treated with an adrenocorticotropic hormone - Acthar Gel for their active SLE.This report is a retrospective study from 5 academic centers describing the effectiveness of the gel in 9 patients with SLE. This begs the question how was the study initiated?The article should really be limited to 8 patients as Patient 9 was lost to follow up and therefore did not satisfy the described inclusion criteria.The authors list the limitations of their study in the discussion.The following are questions that arise from the paperWhat and how many patients were excluded from the study as not meeting study criteria. In other words the patients included all seemed to do well on the Acthar Gel, however were there patients who were on the gel for less than 6 months (thereby not satisfying inclusion criteria ) who did not do well. What were the actual doses of the gel - a mean dose is listed without any standard deviation. As this is a retrospective study the listing of improvement, especially the definition of 50% improvement would have to be a guess as this would not necessarily be recorded in the patient's chart. While it is a small sample size the statistics should include a mean and standard deviation. This is especially the case when the authors list the improvement of the SLEDAI 2K decreasing 3.5, what is this decrease from? There is one significant outlier a sick patient with an initial SLEDAI of 16 who after using the Acthar Gel reduced the SLEDAI to 4, although she was the only one whose arthritis did not improve on Acthar Gel administration. What is the real benefit from using this medication. After all one is merely changing from one type of steroid to another. Is the benefit the ease of administration, less side effects or less long term complicationsIt will be of interest to see the results of the current clinical trials in SLE nephritis, in the 2 patients in this study with nephritis the Acthar Gel was either ineffective or its effects wore off .", "responses": [ { "c_id": "1824", "date": "24 Feb 2016", "name": "Joyce Hui-Yuen", "role": "Author Response", "response": "Thank you very much for your review. We have submitted a new version and include the following in response to your comments:Comment: What and how many patients were excluded from the study as not meeting study criteria. In other words the patients included all seemed to do well on the Acthar Gel, however were there patients who were on the gel for less than 6 months (thereby not satisfying inclusion criteria ) who did not do well.Response: We have modified the timecourse to 3-6 months of Acthar Gel treatment to include all 9 patients, and apologize for any confusion.Comment: What were the actual doses of the gel - a mean dose is listed without any standard deviation.Response: We have removed the word 'mean' from the dosing as all patients were on 80 IU subcutaneously biweekly as per prescribing recommendations.Comment: As this is a retrospective study the listing of improvement, especially the definition of 50% improvement would have to be a guess as this would not necessarily be recorded in the patient's chart.Response: The improvement in outcomes was based on the SRI-50, which attempts to capture a 50% improvement in the patient's condition. This was recorded by the treating physician at the treatment site, and did not involve the central site (i.e., Columbia University) which received all surveys for analysis.Comment: While it is a small sample size the statistics should include a mean and standard deviation. This is especially the case when the authors list the improvement of the SLEDAI 2K decreasing 3.5, what is this decrease from? There is one significant outlier a sick patient with an initial SLEDAI of 16 who after using the Acthar Gel reduced the SLEDAI to 4, although she was the only one whose arthritis did not improve on Acthar Gel administration.Response: In the Abstract, we have modified the baseline and 6-month SLEDAI reports to include the standard deviations and ranges. So the decrease to a mean of 3.5 +/- 2.7 was from the mean baseline of 5.8 +/- 5.0.Comment: What is the real benefit from using this medication. After all one is merely changing from one type of steroid to another. Is the benefit the ease of administration, less side effects or less long term complications.Response: This is an excellent question - we also eagerly await the results of the clinical trials in active SLE, and lupus nephritis patients. At the present time, we have included the following sentence in the last paragraph of the Discussion in an attempt to clarify our conclusion: \"In particular, this study suggests that ACTH may be an attractive alternative to oral corticosteroids in the treatment of lupus; Acthar Gel may improve disease control through steroidogenic as well as direct anti-inflammatory effects, and does not appear to date to carry as severe a side effect profile as corticosteroids.\"Thank you again for your time and critical comments. We very much appreciate your review." } ] } ]	1	https://f1000research.com/articles/4-1103
https://f1000research.com/articles/5-212/v1	24 Feb 16	{ "type": "Opinion Article", "title": "Phytoglobin: a novel nomenclature for plant globins accepted by the globin community at the 2014 XVIII conference on Oxygen-Binding and Sensing Proteins", "authors": [ "Robert Hill", "Mark Hargrove", "Raúl Arredondo-Peter", "Robert Hill", "Mark Hargrove" ], "abstract": "Hemoglobin (Hb) is a heme-containing protein found in the red blood cells of vertebrates. For many years, the only known Hb-like molecule in plants was leghemoglobin (Lb). The discovery that other Hb-like proteins existed in plants led to the term “nonsymbiotic Hbs (nsHbs)” to differentiate them from the Lbs. While this terminology was adequate in the early stages of research on the protein, the complexity of the research in this area necessitates a change in the definition of these proteins to delineate them from red blood cell Hb. At the 2014 XVIII Conference on Oxygen-Binding and Sensing Proteins, the group devoted to the study of heme-containing proteins, this issue was discussed and a consensus was reached on a proposed name change. We propose Phytoglobin (Phytogb) as a logical, descriptive name to describe a heme-containing (Hb-like) protein found in plants. It will be readily recognized by the research community without a prolonged explanation of the origin of the term. The classification system that has been established can essentially remain unchanged substituting Phytogb in place of nsHb. Here, we present a guide to the new nomenclature, with reference to the existing terminology and a phylogenetic scheme, placing the known Phytogbs in the new nomenclature.", "keywords": [ "Algae", "angiosperms", "bryophytes", "gymnosperms", "legumes", "nonsymbiotic", "truncated" ], "content": "\n\nHemoglobin (Hb) is a heme-containing protein found in the red blood cells of vertebrates1. Hemoglobin-like proteins are also found in other tissues of vertebrates where they are given tissue-specific names that help to identify their locations and distinguish them from red blood cell Hb2,3. For many years, the only known Hb-like molecule in plants was leghemoglobin (Lb), a protein induced as a result of the symbiotic relationship between legume plants and nitrogen-fixing bacteria4. The discovery that other Hb-like proteins existed in plants not capable of symbiotic relationships led to the term “nonsymbiotic Hbs (nsHbs)” to differentiate them from the Lbs5. While this terminology was adequate in the early stages of research on the protein, the complexity of the research in this area necessitates a change in the definition of these proteins to delineate them from red blood cell Hb, in keeping with the terminology for other Hb-like proteins, such as myoglobin in muscle, neuroglobin in neuron tissue and cytoglobin in vertebrate cell cytoplasm2,3. In 2001 Hunt et al.6 classified plant Hbs as globin (GLB)0, GLB1, GLB2, GLb3 and GLBS corresponding to undetermined (mostly liverwort and moss) nsHbs, angiosperm nsHbs class/type 1 and nsHbs class/type 2, truncated Hbs and symbiotic Hbs (which included Lbs), respectively. However, an epithet for plant Hbs was absent in this nomenclature and distinctive characteristics for each category were not fully defined resulting in an incomplete classification system.\n\nAt the 2014 XVIII Conference on Oxygen-Binding and Sensing Proteins, the group devoted to the study of heme-containing proteins, the above issue was discussed and a consensus was reached on a proposed name change. Phytoglobin (phyto, plant; globin, heme-containing protein folding structurally similar to the sperm whale myoglobin structure whose heme-Fe is invariably coordinated at the proximal site by His F8), abbreviated as Phytogb, was proposed as a logical, descriptive name to describe a heme-containing (Hb-like) protein found in plants. It will be readily recognized by the research community without a prolonged explanation of the origin of the term, as is the case for ‘nonsymbiotic hemoglobin’. The classification system that has been established can essentially remain unchanged substituting Phytogb in place of nsHb. A guide to the new nomenclature, with reference to the existing terminology, is given in Table 1. A more detailed phylogenetic scheme, placing the known Phytogbs in the new nomenclature, is shown in Figure 1. Also, we propose that acronym for the species-specific Phytogbs corresponds to the first three binomial (i.e. genus and species) letters followed by the Phytogb type and phytogb number of copy. For example, the acronym for rice (Oryza sativa) Phytogb1.1 (see Table 1) corresponds to OrysatPhytogb1.1.\n\naNumerical classification corresponds to that previously proposed by Hunt et al.6. Proteins coded by multiple phytogb gene copy numbers within the same plant species should be indicated as the number of copy after the Phytogb numerical classification. For example, rice (Oryza sativa) Phytogbs 1 and 2 (corresponding to the former nsHbs-1) should be indicated as rice Phytogb1.1 and Phytogb1.2, respectively (see text for a description on the species-specific Phytogbs acronym).\n\nbHeme-Fe coordination and affinity for O2 correspond to those from moss Phytogb07–9, barley10, rice11 and Arabidopsis12 Phytogb1, Arabidopsis Phytogb212, Casuarina SymPhytogb13, soybean Lb14,15 and Arabidopsis Phytogb316 representative of Phytogb0, Phytogb1, Phytogb2, SymPhytogb, Lb and Phytogb3, respectively.\n\ncAmino acid sequence of algal globins analyzed so far17–19 is similar to that of land plant Phytogb0 and Phytogb3, hence algal globins can be classified as Phytogb0 or Phytogb3, respectively.\n\ndSome SymPhytogbs and Lbs (such as the Parasponia20 and Casuarina21 and Chamaecrista22 globins, respectively) are intermediate between Phytogbs1 and Phytogbs2 and SymPhytogbs and Lbs22,23 because they exhibit amino acid sequence similarity to Phytogbs1 and Phytogbs2 (Figure 1) and are localized in non-legume an legume nodules and apparently play a role in symbiotic N2-fixation.\n\nNote that Parasponia, Casuarina, Alnus and Myrica SymPhytogbs are intermediate between SymPhytogbs and Phytogbs1 and Phytogbs2 (see Table 1 for explanation). Figure modified from Garrocho-Villegas et al.23 (reprinted with permission).", "appendix": "Author contributions\n\n\n\nRDH conceived the proposal. RDH, MSH and RAP discussed and proposed the novel nomenclature, prepared the first draft of the manuscript, revised 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\nAuthors wish to express their gratitude to Itzel Rojas Sánchez, Gisselle A. Fuentes and Gustavo Rodríguez-Alonso for evaluating the Phytoglobins nomenclature presented here and providing useful comments.\n\n\nReferences\n\nDickerson RE, Geis I: Hemoglobin: structure, function, evolution, and pathology. Menlo Park, California: The Benjamin/Cummings Pub. Co., Inc.; 1983; 176. Reference Source\n\nVinogradov SN, Hoogewijs D, Bailly X, et al.: A phylogenomic profile of globins. BMC Evol Biol. 2006; 6: 31. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWeber RE, Vinogradov SN: Nonvertebrate hemoglobins: functions and molecular adaptations. Physiol Rev. 2001; 81(2): 569–628. PubMed Abstract\n\nAppleby CA: Leghemoglobin and Rhizobium respiration. Annu Rev Plant Physiol. 1984; 35: 443–478. Publisher Full Text\n\nBogusz D, Appleby CA, Landsmann J, et al.: Functioning haemoglobin genes in non-nodulating plants. Nature. 1988; 331(6152): 178–180. PubMed Abstract \| Publisher Full Text\n\nHunt PW, Watts RA, Trevaskis B, et al.: Expression and evolution of functionally distinct haemoglobin genes in plants. Plant Mol Biol. 2001; 47(5): 677–692. PubMed Abstract \| Publisher Full Text\n\nGarrocho-Villegas V, Arredondo-Peter R: Molecular cloning and characterization of a moss (Ceratodon purpureus) non-symbiotic hemoglobin provides insight into the early evolution of plant non-symbiotic hemoglobins. Mol Biol Evol. 2008; 25(7): 1482–1487. PubMed Abstract \| Publisher Full Text\n\nSmagghe BJ, Kundu S, Hoy JA, et al.: Role of phenylalanine B10 in plant non-symbiotic hemoglobins. Biochemistry. 2006; 45(32): 9735–9745. PubMed Abstract \| Publisher Full Text\n\nVázquez-Limón C, Castro-Bustos S, Arredondo-Peter R: Spectroscopic analysis of moss (Ceratodon purpureus and Physcomitrella patens) recombinant non-symbiotic hemoglobins. Commun Integr Biol. 2012; 5(6): 527–530. PubMed Abstract \| Publisher Full Text\n\nDuff SM, Wittenberg JB, Hill RD: Expression, purification, and properties of recombinant barley (Hordeum sp.) hemoglobin. Optical spectra and reactions with gaseous ligands. J Biol Chem. 1997; 272(27): 16746–16752. PubMed Abstract \| Publisher Full Text\n\nArredondo-Peter R, Hargrove MS, Sarath G, et al.: Rice hemoglobins. Gene cloning, analysis, and O2-binding kinetics of a recombinant protein synthesized in Escherichia coli. Plant Physiol. 1997; 115(3): 1259–1266. PubMed Abstract \| Publisher Full Text\n\nTrevaskis B, Watts RA, Andersson SR, et al.: Two hemoglobin genes in Arabidopsis thaliana: the evolutionary origins of leghemoglobins. Proc Natl Acad Sci USA. 1997; 94(22): 12230–12234. PubMed Abstract \| Free Full Text\n\nFleming AI, Wittenberg JB, Wittenberg BA, et al.: The purification, characterization and ligand-binding kinetics of hemoglobins from root nodules of the non-leguminous Casuarina glauca-Frankia symbiosis. Biochim Biophys Acta. 1987; 911(2): 209–220. Publisher Full Text\n\nHargrove MS, Barry JK, Brucker EA, et al.: Characterization of recombinant soybean leghemoglobin a and apolar distal histidine mutants. J Mol Biol. 1997; 266(5): 1032–1042. PubMed Abstract \| Publisher Full Text\n\nWittenberg JB, Appleby CA, Wittenberg BA: The kinetics of the reactions of leghemoglobin with oxygen and carbon monoxide. J Biol Chem. 1972; 247(2): 527–31. PubMed Abstract\n\nWatts RA, Hunt PW, Hvitved AN, et al.: A hemoglobin from plants homologous to truncated hemoglobins of microorganisms. Proc Natl Acad Sci USA. 2001; 98(18): 10119–10124. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFernández I, Vinogradov SN, Arredondo-Peter R: Identification and in silico characterization of a putative ancestor to land plant non-symbiotic hemoglobins from the prasinophyceae algae Micromonas and Ostreococcus. Global J Biochem. 2010; 1: 18–30. Reference Source\n\nVinogradov SN, Fernández I, Hoogewijs D, et al.: Phylogenetic relationships of 3/3 and 2/2 hemoglobins in Archaeplastida genomes to bacterial and other eukaryote hemoglobins. Mol Plant. 2011; 4(1): 42–58. PubMed Abstract \| Publisher Full Text\n\nVinogradov SN, Hoogewijs D, Arredondo-Peter R: What are the origins and phylogeny of plant hemoglobins? Commun Integr Biol. 2011; 4(4): 443–445. PubMed Abstract \| Free Full Text\n\nAppleby CA, Tjepkema JD, Trinick MJ: Hemoglobin in a nonleguminous plant, Parasponia: possible genetic origin and function in nitrogen fixation. Science. 1983; 220(4600): 951–953. PubMed Abstract \| Publisher Full Text\n\nJacobsen-Lyon K, Jensen EO, Jørgensen JE, et al.: Symbiotic and non-symbiotic hemoglobin genes of Casuarina glauca. Plant Cell. 1995; 7(2): 213–223. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGopalasubramaniam SK, Kovacs F, Violante-Mota F, et al.: Cloning and characterization of a caesalpinoid (Chamaecrista fasciculata) hemoglobin: the structural transition from a nonsymbiotic hemoglobin to a leghemoglobin. Proteins. 2008; 72(1): 252–260. PubMed Abstract \| Publisher Full Text\n\nGarrocho-Villegas V, Gopalasubramaniam SK, Arredondo-Peter R: Plant hemoglobins: what we know six decades after their discovery. Gene. 2007; 398(1–2): 78–85. PubMed Abstract \| Publisher Full Text" }	[ { "id": "12635", "date": "08 Mar 2016", "name": "Alexey F. Topunov", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe reviewing article is dedicated to very interesting and actual issue. After proposition of the term “leghemoglobin” (Lb) by Virtanen and Laine (1946) 1 it was used only for hemoglobins (Hb) of the leguminous plants because it was the only one group of plants known to contain Hb. However after observing of Hbs in non-leguminous plants (Appleby et al. 1983 2, Tjepkema 1983 3) the ironic situation originated. “Non-leguminous” hemoglobins are evolutionary closer to leghemoglobins, but according to their denominations they looked to be similar to animal ones. Even hemoglobin of cyanobacteria received its own specific denomination “cyanoglobin” (Hill et al 1996) 4 although it was initially illogically named “Myogloblin in a cyanobacterium” (Potts et al 1992) 5, but plant Hbs were poor relatives in this family. The necessity of new terminology for plant hemoglobins appeared and such thoughts apparented and were discussed. The proposition of the new terminology “phytoglobin” (Phb) was already once stated. It was made in Bach Institute of Biochemistry (Moscow, Russia) in the book by Kretovich 6 and this term was even used in the published article (Topunov 1994) 7. This idea was not further developed at that time and in the next paper (Topunov 1995) 8 the old word “leghemoglobin” was employed again but using of the “phytoglobin” terminology in the article 7 was referred.In conclusion: it is the right desire to revive this terminology for plant hemoglobins and it has to be promoted and continued. There is the one difference between these two propositions of the “phytoglobin” term. In 1990s it was used as the general terminology for all plant hemoglobins (from both leguminous and non-leguminous plants) 6,7 so “leghemoglobin” term could be excluded. Now it is proposed for plant hemoglobins aside from Lb, and it looks more pragmatic because scientists are accustomed to the “leghemoglobin” term and there is no need to exclude it from the scientific practice.", "responses": [ { "c_id": "1856", "date": "09 Mar 2016", "name": "Raul Arredondo-Peter", "role": "Author Response", "response": "We thank Dr. Topunov for his evaluation and providing information that complements the contents of this article." } ] }, { "id": "12637", "date": "11 Mar 2016", "name": "Kurt V Fagerstedt", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe change in the hemoglobin/phytoglobin nomenclature presented in Hill et al. paper is timely and well supported by the hemoglobin and phytoglobin community. The change has been discussed in many conferences including the 2014 XVIII Conference on Oxygen-Binding and Sensing Proteins mentioned in the paper. The system presented in Table 1 is logical and supported by the protein structures and by the phylogenetic tree presented in Figure 2. The only and very slight problem is in the positioning of symbiotic hemoglobins present in non-legume nitrogen fixing plants, which have amino acid sequence similarities with both Phytogbs1 and 2. However, their positioning as a separate group in Table 1 is supported by the fact that they are only found in the nodules of actinorhizal plants.Dr. Topunov has provided a historical perspective on the evolution of the name phytoglobin in his referee report, which clearly shows both the need for the term and no scientific discrepancy in its use. I agree wholeheartedly.", "responses": [ { "c_id": "1862", "date": "14 Mar 2016", "name": "Raul Arredondo-Peter", "role": "Author Response", "response": "We thank Dr. Fagerstedt for evaluating this article and his comments." } ] } ]	1	https://f1000research.com/articles/5-212
https://f1000research.com/articles/5-209/v1	23 Feb 16	{ "type": "Review", "title": "Recent advances in the pathogenesis and treatment of paroxysmal nocturnal hemoglobinuria", "authors": [ "Lucio Luzzatto" ], "abstract": "Paroxysmal nocturnal hemoglobinuria (PNH) is a very rare disease that has been investigated for over one century and has revealed unique aspects of the pathogenesis and pathophysiology of a hemolytic anemia. PNH results from expansion of a clone of hematopoietic cells that, as a consequence of an inactivating mutation of the X-linked gene PIG-A, are deficient in glycosylphosphatidylinositol (GPI)-linked proteins: since these include the surface membrane complement-regulatory proteins CD55 and CD59, the red cells arising from this clone are exquisitely sensitive to lysis by activated complement. Until a decade ago, the treatment options for PNH were either supportive treatment – often including blood transfusion, anti-thrombosis prophylaxis, and sometimes thrombolytic therapy – or allogeneic bone marrow transplantation. Since 2007, PNH has received renewed and much wider attention because a new form of treatment has become available, namely complement blockade through the anti-C5 monoclonal antibody eculizumab. This brief review focuses on two specific aspects of PNH: (1) response to eculizumab, variability of response, and how this new agent has impacted favorably on the outlook and on the quality of life of patients; and (2) with respect to pathogenesis, new evidence supports the notion that expansion of the PNH clone results from T-cell-mediated auto-immune damage to hematopoietic stem cells, with the GPI molecule as target. Indeed, GPI-specific CD8+ T cells – which have been identified in PNH patients – would spare selectively GPI-negative stem cells, thus enabling them to re-populate the marrow of a patient who would otherwise have aplastic anemia.", "keywords": [ "Paroxysmal nocturnal hemoglobinuria", "hemolytic anemia", "clonal disorder", "PNH", "GPI" ], "content": "Introduction\n\nParoxysmal nocturnal hemoglobinuria (PNH) is a unique disorder in more ways than one1. First, it is a hemolytic anemia, but, unlike any other hemolytic anemia, it is frequently associated with pancytopenia. Second, it is a thrombophilic condition in which it is not unusual for thrombosis to take place, paradoxically, in a thrombocytopenic patient. Third, although it is due to an intrinsic abnormality of the red cell, it is an acquired disorder2. This last characteristic feature, a long time ago, led to the notion and then to the demonstration that PNH is a clonal disorder3, and we now know that the clone originates from a hematopoietic stem cell (HSC) with a somatic mutation that inactivates the X-linked gene PIG-A4. This gene encodes one of the subunits of a specific N-acetylglucosamine transferase5, the first enzyme of the complex pathway that leads to the synthesis of the glycosylphosphatidylinositol (GPI) molecule that tethers many proteins to the cell surface6. As a result, the cells belonging to the PNH clone are severely or totally deficient in these proteins: they have a GPI-negative phenotype. Since the two red cell surface complement regulators CD55 and CD59 are GPI-linked proteins7, red cells belonging to a PNH clone are exquisitely sensitive to complement, and they will hemolyze when complement is activated: indeed, in most cases intravascular hemolysis is a dominant pathophysiological feature of PNH8.\n\nIt has been made clear from several animal models that inactivating mutations of PIG-A do not confer to HSCs a selective growth advantage9,10. Indeed, to understand what enables the PIG-A mutant GPI-negative clone to expand has been a challenge. Three hypotheses have been considered: (i) since rare somatic mutations are present in every normal person, and since relatively few HSCs are active in normal hematopoiesis, one PIG-A mutant HSC – even though it has no selective advantage – might simply by chance (genetic drift) produce a large proportion of the peripheral blood cells, and these will be GPI negative11; (ii) there is a close relationship between aplastic anemia (AA) and PNH, and it has been suggested that this is anything but a coincidence; the cell-mediated auto-immune attack that is believed to cause AA may spare selectively GPI-negative HSCs: in other words, the bone marrow environment prevailing in PNH patients creates for the PIG-A mutant GPI-negative clone a growth advantage which is not intrinsic but is conditional on the environment12; (iii) in the PIG-A mutant clone, there may be additional mutation(s) that confer to the respective subclone(s) an intrinsic growth advantage. In two patients with PNH, a mutation of the HMGA2 gene may have played this role13, and HMGA2 over-expression has been reported in additional cases14. In addition, a recent massive parallel sequencing study, while confirming that the only gene mutated in all cases is PIG-A, has revealed mutations in several other genes, some of them already known to be mutated in myelodysplastic syndromes15.\n\nThere are many published reviews on PNH and here we intend to focus only on selected recent developments.\n\n\nNovel therapy\n\nThe last decade has been marked by the clinical trials of eculizumab (ECU)16,17, followed in 2007 by the prompt introduction of this agent into standard therapy. ECU is a humanized monoclonal antibody specific for the human plasma complement component C5: by binding to C5, ECU blocks the distal complement pathway and thus protects PNH red cells from complement-mediated lysis. In patients with PNH, anemia per se causes fatigue; at the same time, intravascular hemolysis often entails unpleasant and sometimes severe symptoms, such as abdominal pain, dysphagia, and erectile dysfunction. ECU terminates intravascular hemolysis in virtually all patients: as a result, these symptoms are abrogated, and fatigue from anemia is often alleviated. Up to two-thirds of patients who were transfusion dependent become transfusion independent18. It is not an exaggeration to say that ECU has changed the life of many PNH patients.\n\n\nThe spectrum of response to eculizumab and pharmacogenetics\n\nWith respect to the response to ECU, we must consider at least two different end-points: (a) inhibition of intravascular hemolysis consequent on complement blockade and (b) overall clinical benefit. The cases where response to ECU fails to meet end-point (a) are very rare: they have been reported in patients from Japan who have a specific mutation in the C5 gene, which entails in the C5 protein an Arg885His amino acid replacement, which in turn prevents binding of ECU19. This mutation is polymorphic in Japan (heterozygote frequency of about 3.5%) and in China, but to date it has not been observed elsewhere.\n\nIn contrast to (a), which behaves like an all-or-none end-point, clinical benefit (b) is a continuous variable. Of those patients who become transfusion independent, some experience a net increase in hemoglobin level as well (see Figure 1); others stay on more or less that same hemoglobin level to retain what they previously needed transfusion support for18. Some patients (about 25% in our experience) still need recurrent blood transfusion (usually at less frequent intervals than before); however, even these patients report improved quality of life because they no longer experience the unpleasant subjective symptoms listed above.\n\nThis variability of response (Figure 1) is not yet fully explained. In some patients, a poor or suboptimal response may indicate that the extent to which bone marrow failure was contributing to anemia had been under-estimated. In others, a suboptimal response is related at least in part to an important shift in pathophysiology that takes place in patients on ECU (see Figure 2). In essence, because GPI-negative (PNH) red cells are protected from complement lysis, they survive much longer in circulation (at first sight regarded as paradoxical, an increase in the proportion of PNH red cells is a typical feature of patients on ECU). ECU affects only the distal complement pathway, thus neutralizing the handicap caused by the deficiency of CD59 on PNH red cells, but since these are also deficient in CD55, there will be a gradual accumulation of C3 fragments on their surface (see Figure 2), and these C3-opsonized red cells will be susceptible to phagocytosis by macrophages in the reticuloendothelial system20,21. This mechanism of extravascular hemolysis – which we must regard as iatrogenic – causes the Coombs test to become positive22 (it is classically negative in untreated PNH); more importantly, it may limit the improvement in anemia because it develops at the same time that intravascular hemolysis ceases.\n\nIn untreated patients (upper cartoon), as soon as C3 (yellow circles) is bound to a GPI-negative red cell (lacking CD55), C5 will be activated, the membrane attack complex (MAC) will form, and the red cell (which also lacks CD59) will be lyzed: accordingly, no red cells with C3 bound are seen in the upper flow cytometry pattern on the left. In patients on eculizumab (lower cartoon), with C5 blocked, no MAC is formed and there is no lysis; in return, a much larger number of C3 molecules accumulate (see lower flow cytometry pattern on the left); thus, the red cell is opsonized and will be prey to macrophages. This is the likely mechanism of extravascular hemolysis in PNH patients on eculizumab (modified from 18).\n\nAt least one factor that influences markedly the development of this consequence of ECU treatment is, once again, genetic and complement related. When patients on ECU are stratified according to their genotype for the complement receptor gene CR1, it is seen that among those who have a suboptimal hematological response, homozygotes for the low-affinity allele L are markedly more common, and homozygotes for the high-affinity allele H much rarer than expected; heterozygotes are intermediate, indicating a dosage effect23.\n\n\nThrombosis in paroxysmal nocturnal hemoglobinuria\n\nVenous thrombosis, particularly in abdominal veins or in intracranial veins, remains one of the most feared complications of PNH. Although the literature on this subject is vast, the mechanism that makes PNH the most vicious acquired thrombophilic state known to medicine18 remains elusive. Thromboplastin-like substances from hemolyzed red cells, inappropriate platelet activation, and failure of fibrinolysis may all play a role24. But in recent years the most significant change has been caused, also in this area, by the use of ECU. Although there has been no formal trial on the impact of ECU on thrombosis, patients on ECU have some changes in laboratory parameters of hemostasis25, and they have fewer thrombotic episodes than one might have expected from previous experience26,27. This is obviously of great clinical importance; in addition, it indicates that complement blockade (whether it acts via red cells or in any other way) does protect from thrombosis, although not completely because thrombosis on ECU can still occur27.\n\nAt any rate, the risk of thrombosis is still highly relevant to the management of PNH patients for several reasons. First, to many patients living in many parts of the world, ECU is not available, mainly because it is too expensive: for these patients, it is important to have a clear policy with respect to prophylaxis of thrombosis (for example, see 18). For these patients, it is likely that coumadin will be replaced soon by new oral anticoagulants (NOACs), although there is as yet little experience in PNH with these agents. Second, patients with PNH who also have an inherited thrombophilic state (e.g. a prothrombin mutation or an anti-thrombin III defect) are at even greater risk than the others and one must consider, even when they are already on ECU, having them on anticoagulants as well. Third, patients not previously diagnosed with PNH may present with recent thrombosis: in these cases, it would seem reasonable to introduce ECU immediately, but if this is not possible one ought to consider thrombolytic therapy with tissue plasminogen activator (tPA), which can be highly effective28. Fourth, in some PNH patients, a special problem, almost invariably secondary to splenic vein and/or portal vein thrombosis, is splenomegaly with hypersplenism, which may cause cytopenias or make them worse: in such cases, selective splenic artery embolization (SSAE), carried out in steps, will reduce spleen size and alleviate cytopenias29. An extra bonus of this procedure is that when in a patient on ECU extravascular hemolysis is severe, it may be relieved by SSAE29,30, a safer alternative compared to splenectomy31.\n\n\nThe mechanism of clonal expansion\n\nAs mentioned in the introduction, in order to account for the expansion of a GPI-negative blood cell population in patients with PNH, one possible explanation is a selective environment. The selective process must be able to differentiate stem cells that are GPI positive from those that are GPI negative, even if they are otherwise undistinguishable32. Cells of the immune system are specialized in recognizing chemical structures, and therefore they are good candidates for being the agents of highly sophisticated selection. The immune system is already presumed to be involved in the pathogenesis of idiopathic AA33, and although the phrase AA-PNH syndrome was coined perhaps with the idea that this was a peculiar subtype of PNH34, in our experience and in that of others35, a history of more or less severe AA at the time of diagnosing PNH is present in at least one-half of all cases. It may perhaps soon become the rule rather than the exception. This is consistent with the notion that expansion of a GPI-negative clone, characteristic of PNH, is also immune mediated.\n\nIn principle, the target of immune selection might be either (i) a GPI-linked protein – peptides from which would be presented by the major histocompatibility complex (MHC) – or (ii) the GPI molecule itself (see Figure 3), which would be presented not by the MHC but by the structurally related molecule CD1d36. The former possibility was not supported by the finding that the amino acid sequence of the CD3 of the βeta chain of the T cell receptor of selected CD57+ T cells was identical in PNH patients who had different HLA types37. On the other hand, when CD8+ T cells were co-cultured with HLA-negative antigen-presenting B cells previously engineered to express CD1d, significantly higher levels of reactive T cells (producing interferon gamma were found in PNH patients compared to normal controls38. When the antigen-presenting cells were further engineered to lose competence for endogenous synthesis of GPI, this reactivity became strictly dependent on loading their surface CD1d with exogenous human GPI produced by organic synthesis39. Similar results were obtained with autologous antigen-presenting cells from peripheral blood monocytes from the same patients. These data provide the first direct evidence for the presence of GPI-reactive CD1d-restricted T cells in PNH patients38.\n\nThe diagram shows, on the right-hand side, the presumed pathogenesis of aplastic anemia (AA): a T-cell-mediated autoimmune attack damages hematopoietic stem cells (HSCs). The target of the auto-reactive T cells may be the glycosylphosphatidylinositol (GPI) molecule or another molecule expressed on HSCs. On the left-hand side, an inactivating mutation of the PIG-A gene in a HSC produces a GPI-negative hematopoietic clone. In the absence of the PIG-A mutant clone, the autoimmune attack, even if GPI is targeted, produces AA. In the absence of the autoimmune attack, a PIG-A mutant clone will be of no consequence (subclinical). Only if both a PIG-A mutant clone and a GPI-targeted autoimmune attack co-exist will the mutant clone expand and cause clinical PNH.", "appendix": "Competing interests\n\n\n\nThe author declares that he has 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\nDacie JV: Paroxysmal nocturnal haemoglobinuria. The Haemolytic Anaemias: Drug and Chemical Induced Haemolytic Anaemias, Paroxysmal Nocturnal Haemglobinuria, and Haemolytic Disease of the Newborn. London: Churchill Livingston; 1999; 139–330.\n\nLuzzatto L: Paroxysmal nocturnal haemoglobinuria. In: Warrel DA, Cox TM, Firth JD, editors. Oxford Textbook of Medicine. 5th ed. Oxford (UK): Oxford University Press; 2010; 4298–302. Publisher Full Text\n\nOni SB, Osunkoya BO, Luzzatto L: Paroxysmal nocturnal hemoglobinuria: evidence for monoclonal origin of abnormal red cells. Blood. 1970; 36(2): 145–52. PubMed Abstract\n\nTakeda J, Miyata T, Kawagoe K, et al.: Deficiency of the GPI anchor caused by a somatic mutation of the PIG-A gene in paroxysmal nocturnal hemoglobinuria. Cell. 1993; 73(4): 703–11. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nMiyata T, Takeda J, Iida Y, et al.: The cloning of PIG-A, a component in the early step of GPI-anchor biosynthesis. Science. 1993; 259(5099): 1318–20. PubMed Abstract \| Publisher Full Text\n\nKinoshita T, Fujita M, Maeda Y: Biosynthesis, remodelling and functions of mammalian GPI-anchored proteins: recent progress. J Biochem. 2008; 144(3): 287–94. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nBoccuni P, Del Vecchio L, Di Noto R, et al.: Glycosyl phosphatidylinositol (GPI)-anchored molecules and the pathogenesis of paroxysmal nocturnal hemoglobinuria. Crit Rev Oncol Hematol. 2000; 33(1): 25–43. PubMed Abstract \| Publisher Full Text\n\nLuzzatto L, Gianfaldoni G: Recent advances in biological and clinical aspects of paroxysmal nocturnal hemoglobinuria. Int J Hematol. 2006; 84(2): 104–12. PubMed Abstract \| Publisher Full Text\n\nRosti V: Murine models of paroxysmal nocturnal hemoglobinuria. Ann N Y Acad Sci. 2002; 963: 290–6. PubMed Abstract \| Publisher Full Text\n\nLuzzatto L: Paroxysmal murine Hemoglobinuria(?): A model for human PNH. Blood. 1999; 94(9): 2941–4. PubMed Abstract\n\nDingli D, Luzzatto L, Pacheco JM: Neutral evolution in paroxysmal nocturnal hemoglobinuria. Proc Natl Acad Sci U S A. 2008; 105(47): 18496–500. PubMed Abstract \| Publisher Full Text\n\nRotoli B, Luzzatto L: Paroxysmal nocturnal hemoglobinuria. Semin Hematol. 1989; 26(3): 201–7. PubMed Abstract\n\nInoue N, Izui-Sarumaru T, Murakami Y, et al.: Molecular basis of clonal expansion of hematopoiesis in 2 patients with paroxysmal nocturnal hemoglobinuria (PNH). Blood. 2006; 108(13): 4232–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMurakami Y, Inoue N, Shichishima T, et al.: Deregulated expression of HMGA2 is implicated in clonal expansion of PIGA deficient cells in paroxysmal nocturnal haemoglobinuria. Br J Haematol. 2012; 156(3): 383–7. PubMed Abstract \| Publisher Full Text\n\nShen W, Clemente MJ, Hosono N, et al.: Deep sequencing reveals stepwise mutation acquisition in paroxysmal nocturnal hemoglobinuria. J Clin Invest. 2014; 124(10): 4529–38. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHillmen P, Hall C, Marsh JC, et al.: Effect of eculizumab on hemolysis and transfusion requirements in patients with paroxysmal nocturnal hemoglobinuria. N Engl J Med. 2004; 350(6): 552–9. PubMed Abstract \| Publisher Full Text\n\nHillmen P, Young NS, Schubert J, et al.: The complement inhibitor eculizumab in paroxysmal nocturnal hemoglobinuria. N Engl J Med. 2006; 355(12): 1233–43. PubMed Abstract \| Publisher Full Text\n\nLuzzatto L, Gianfaldoni G, Notaro R: Management of paroxysmal nocturnal haemoglobinuria: a personal view. Br J Haematol. 2011; 153(6): 709–20. PubMed Abstract \| Publisher Full Text\n\nNishimura J, Yamamoto M, Hayashi S, et al.: Genetic variants in C5 and poor response to eculizumab. N Engl J Med. 2014; 370(7): 632–9. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nRisitano AM, Notaro R, Marando L, et al.: Complement fraction 3 binding on erythrocytes as additional mechanism of disease in paroxysmal nocturnal hemoglobinuria patients treated by eculizumab. Blood. 2009; 113(17): 4094–100. PubMed Abstract \| Publisher Full Text\n\nLin Z, Schmidt CQ, Koutsogiannaki S, et al.: Complement C3dg-mediated erythrophagocytosis: implications for paroxysmal nocturnal hemoglobinuria. Blood. 2015; 126(7): 891–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHöchsmann B, Leichtle R, von Zabern I, et al.: Paroxysmal nocturnal haemoglobinuria treatment with eculizumab is associated with a positive direct antiglobulin test. Vox Sang. 2012; 102(2): 159–66. PubMed Abstract \| Publisher Full Text\n\nRondelli T, Risitano AM, Peffault de Latour R, et al.: Polymorphism of the complement receptor 1 gene correlates with the hematologic response to eculizumab in patients with paroxysmal nocturnal hemoglobinuria. Haematologica. 2014; 99(2): 262–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHill A, Kelly RJ, Hillmen P: Thrombosis in paroxysmal nocturnal hemoglobinuria. Blood. 2013; 121(25): 4985–96; quiz 5105. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nHelley D, de Latour RP, Porcher R, et al.: Evaluation of hemostasis and endothelial function in patients with paroxysmal nocturnal hemoglobinuria receiving eculizumab. Haematologica. 2010; 95(4): 574–81. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHillmen P, Muus P, Dührsen U, et al.: Effect of the complement inhibitor eculizumab on thromboembolism in patients with paroxysmal nocturnal hemoglobinuria. Blood. 2007; 110(12): 4123–8. PubMed Abstract \| Publisher Full Text\n\nKelly RJ, Hill A, Arnold LM, et al.: Long-term treatment with eculizumab in paroxysmal nocturnal hemoglobinuria: sustained efficacy and improved survival. Blood. 2011; 117(25): 6786–92. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nAraten DJ, Notaro R, Thaler HT, et al.: Thrombolytic therapy is effective in paroxysmal nocturnal hemoglobinuria: a series of nine patients and a review of the literature. Haematologica. 2012; 97(3): 344–52. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nAraten DJ, Iori AP, Brown K, et al.: Selective splenic artery embolization for the treatment of thrombocytopenia and hypersplenism in paroxysmal nocturnal hemoglobinuria. J Hematol Oncol. 2014; 7: 27. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKrishnan SK, Hill A, Hillmen P, et al.: Improving cytopenia with splenic artery embolization in a patient with paroxysmal nocturnal hemoglobinuria on eculizumab. Int J Hematol. 2013; 98(6): 716–8. PubMed Abstract \| Publisher Full Text\n\nRisitano AM, Marando L, Seneca E, et al.: Hemoglobin normalization after splenectomy in a paroxysmal nocturnal hemoglobinuria patient treated by eculizumab. Blood. 2008; 112(2): 449–51. PubMed Abstract \| Publisher Full Text\n\nLuzzatto L, Bessler M, Rotoli B: Somatic mutations in paroxysmal nocturnal hemoglobinuria: a blessing in disguise? Cell. 1997; 88(1): 1–4. PubMed Abstract \| Publisher Full Text\n\nYoung NS, Maciejewski J: The pathophysiology of acquired aplastic anemia. N Engl J Med. 1997; 336(19): 1365–72. PubMed Abstract \| Publisher Full Text\n\nLewis SM, Dacie JV: The aplastic anaemia--paroxysmal nocturnal haemoglobinuria syndrome. Br J Haematol. 1967; 13(2): 236–51. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nSchrezenmeier H, Muus P, Socié G, et al.: Baseline characteristics and disease burden in patients in the International Paroxysmal Nocturnal Hemoglobinuria Registry. Haematologica. 2014; 99(5): 922–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChiu YH, Jayawardena J, Weiss A, et al.: Distinct subsets of CD1d-restricted T cells recognize self-antigens loaded in different cellular compartments. J Exp Med. 1999; 189(1): 103–10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGargiulo L, Lastraioli S, Cerruti G, et al.: Highly homologous T-cell receptor beta sequences support a common target for autoreactive T cells in most patients with paroxysmal nocturnal hemoglobinuria. Blood. 2007; 109(11): 5036–42. PubMed Abstract \| Publisher Full Text\n\nGargiulo L, Papaioannou M, Sica M, et al.: Glycosylphosphatidylinositol-specific, CD1d-restricted T cells in paroxysmal nocturnal hemoglobinuria. Blood. 2013; 121(14): 2753–61. PubMed Abstract \| Publisher Full Text\n\nRichichi B, Luzzatto L, Notaro R, et al.: Synthesis of the essential core of the human glycosylphosphatidylinositol (GPI) anchor. Bioorg Chem. 2011; 39(2): 88–93. PubMed Abstract \| Publisher Full Text" }	[ { "id": "12622", "date": "23 Feb 2016", "name": "Peter Hillmen", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12623", "date": "23 Feb 2016", "name": "Anita Hill", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-209
https://f1000research.com/articles/5-208/v1	23 Feb 16	{ "type": "Review", "title": "Recent Advances in Understanding and Engineering Polyketide Synthesis", "authors": [ "Wenjun Zhang", "Joyce Liu", "Joyce Liu" ], "abstract": "Polyketides are a diverse group of natural products that form the basis of many important drugs. The engineering of the polyketide synthase (PKS) enzymes responsible for the formation of these compounds has long been considered to have great potential for producing new bioactive molecules. Recent advances in this field have contributed to the understanding of this powerful and complex enzymatic machinery, particularly with regard to domain activity and engineering, unique building block formation and incorporation, and programming rules and limitations. New developments in tools for in vitro biochemical analysis, full-length megasynthase structural studies, and in vivo heterologous expression will continue to improve our fundamental understanding of polyketide synthesis as well as our ability to engineer the production of polyketides.", "keywords": [ "Polyketides", "polyketide synthesis", "polyketide synthase" ], "content": "Introduction\n\nPolyketide compounds are a large family of natural products with great structural diversity and complexity. Many of these compounds are valued for their potent biological activities, and particularly well-known examples include erythromycin, tetracycline, rifamycin, and lovastatin. Polyketides are formed by a family of enzymes known as polyketide synthases (PKSs), which often operate in an assembly line-like fashion to join together acyl coenzyme A (CoA) building blocks1. The core catalytic domains of type I and II PKSs include the ketosynthase (KS) domain, which is responsible for catalyzing decarboxylative Claisen condensations for chain extension; the acyltransferase (AT) domain, which is responsible for building block selection and loading; and the acyl carrier protein (ACP) domain, on which the polyketide chain is elongated (Figure 1A). Additional enzymes that may be either part of the PKS megasynthase or standalone can modify the nascent polyketide chain during or post assembly, and these enzymes further contribute to the diversity and complexity of polyketides that can be produced.\n\n(A) Scheme showing crotonyl-CoA carboxylase/reductase (CCR)-catalyzed biosynthesis of unusual extender units and their subsequent incorporation into polyketide scaffolds. Possible R’ groups include aliphatics, alkyl halides, and aromatics; R represents the donor polyketide intermediate. (B) Structures of novel polyketides with the modifications from atypical building blocks shown in red.\n\nBecause of the inherent modularity of many PKSs and their vast potential for producing pharmaceutically relevant compounds, there has been longstanding interest in engineering these enzymes to produce novel polyketides in a predictable manner. Decades of work ranging from the characterization of PKSs with unique attributes to the fundamental understanding of how these enzymes function to the manipulation of catalytic domains and modules to generate unnatural products have brought us closer to this goal (Figure 1B)2,3. In this short commentary, we discuss recent advances in these areas, focusing on both the new knowledge about PKSs that has been garnered and the tools that have been developed to facilitate efforts in PKS engineering. While the body of work on PKSs is vast, we have restricted our discussion here to a few selected themes found in PKS research from the past couple of years.\n\n\nRecent advances in understanding polyketide synthesis\n\nAs the “gatekeeping” domain, the AT domain has been a key target for polyketide engineering, and a number of recent studies continue to focus on understanding and engineering building block selection by this domain, particularly ATs that recognize atypical substrates, i.e. substrates other than malonyl-CoA or methylmalonyl-CoA. For example, an AT domain from the monensin PKS4 and the loading AT from the avermectin PKS5, both with relaxed substrate specificities, were subjected to computational modeling and structural analysis, respectively, to enable the identification of residues responsible for substrate binding and specificity. Similarly, the AT domain of ZmaA, which specifically recognizes the precursor hydroxymalonyl-ACP, was investigated through structural and biochemical analysis, and this study showed that the donor carrier protein itself biased extender unit selection6. Based on the promiscuity of the loading AT and a mutation in one of the extending ATs of 6-deoxyerythronolide B synthase (DEBS), short-chain alkynoic building blocks have also been incorporated into the backbone of erythromycin (Figure 1B)7,8. In addition, several other recent studies have centered on trans-ATs, which may be easier to engineer than ATs acting in cis. Through in vitro kinetic studies, it was shown that although trans-ATs can complement inactivated cis-ATs, which can be a useful approach for introducing alternative extender units into polyketides, the efficiency of trans-AT complementation is greatly affected by the identity of both the carboxyacyl-CoA and the ACP substrates9. Promisingly, several key electrostatic interactions that define the interaction epitope between a trans-AT promiscuous to carboxyacyl-CoA substrates and its cognate ACP were identified, and this knowledge was further leveraged to engineer a noncognate ACP into a detectable substrate for this trans-AT via the introduction of a single amino acid substitution10. In addition, a trans-AT has also been used to site-selectively incorporate fluorine into a polyketide backbone11.\n\nAside from these AT domain studies, there have also been studies focused on the biosynthetic aspect of unique PKS building blocks. In particular, promiscuous malonyl-CoA synthetase variants have been used to synthesize a broad range of malonyl-CoA extender units substituted at the C2 position12,13. In addition, a family of crotonyl-CoA carboxylase/reductase (CCR) enzymes that transform α,β-unsaturated acyl-CoA substrates to the corresponding carboxyacyl-CoA extender units have also received great attention. CCR enzymes are typically quite flexible and have already been demonstrated to generate both aliphatic CoA- and aromatic CoA-linked extender units (Figure 1A)14–17. The recent structure-based engineering of a CCR enzyme from antimycin biosynthesis afforded the production of several new polyketide extender units18. This work also demonstrated the first use of heterocyclic and substituted arene extender units by PKS machinery (Figure 1B), though it is notable that not all of the generated α-substituted malonyl-CoAs could be accepted by the PKS. In addition to CoA building blocks, dedicated ACP-dependent pathways are often found to generate atypical building blocks for PKSs as well. One example is our recently elucidated ACP-dependent terminal alkyne biosynthetic machinery, which was further exploited for the in situ generation and incorporation of terminal alkynes into polyketide scaffolds as alkynoic starter or extender units (Figure 1B)19. This work demonstrates the feasibility of de novo biosynthesis of terminal alkyne-tagged polyketides that can be subjected to in situ biorthogonal chemistry for further modification. Another recent example is the DH-KR (dehydratase-ketoreductase) bifunctional proteins that were characterized to convert glyceryl-S-ACP into the unusual lactyl-S-ACP starter unit in FR901464 and lankacidin biosynthesis20.\n\nAnother area of focus in recent PKS understanding and engineering lies in iterative type I PKSs, which are typically found in fungi, though more examples are now emerging from bacteria. Although numerous bioactive polyketides are synthesized by iterative type I PKSs, unlike the well-known linear assembly line of multi-modular type I PKSs, these fungal PKSs have only one module that is used iteratively, and the related programming rules regarding substrate selection, catalytic domain utilization in each elongation cycle, regiospecific modification, polyketide chain length control, chain release and transfer, etc. are only just beginning to be understood21,22. For the relatively simple class of non-reducing iterative type I PKSs (NR-PKSs) that are involved in aromatic polyketide synthesis, the starter unit ACP transacylase (SAT) domain has been the focus of several recent studies including domain swapping to generate new aromatic polyketides23 and structural analysis to identify the basis for acyl unit selection24, since unnatural starter units are often properly processed by the rest of the catalytic domains of NR-PKSs (Figure 1B)25. In addition, systematic in vitro domain swapping of NR-PKSs followed by examination of the resulting on-target and shunt products highlighted the important effects of chain length control by KS domains, editing by thioesterase (TE) domains, and inter-domain interactions on combinatorial biosynthesis26. While some additional work has also been done on highly and partially reducing PKSs27,28, particularly with regards to combinatorialization with NR-PKSs29 or nonribosomal peptide synthetases (NRPSs)30, the relative lack of understanding about the programming of these types of PKSs makes them more difficult to engineer effectively.\n\n\nRecent developments in tools for understanding and engineering PKSs\n\nAs polyketide engineering continues to progress, the development of tools and strategies to understand the underlying PKS programming rules and exploit them for the diversification and overproduction of new polyketide compounds is of paramount importance. In this vein, in vitro biochemical analysis using purified enzymes continues to serve as the most important method for studying PKS enzymology, particularly in understanding the precise function and substrate specificity of catalytic domains, reaction mechanisms, and the internal kinetics of the catalytic program. Despite recent significant improvements in the sensitivity and accuracy of mass spectrometry31, it remains challenging to directly detect and quantify the majority of ACP-bound biosynthetic intermediates of polyketides. Alternatively, total enzymatic reconstitution of polyketide synthesis in vitro serves as one of the most common assay methods for biochemical analysis16,32,33. Additionally, several unique quantitative and facile assay strategies have been developed recently; of notable interest are those coupling PKS-catalyzed reactions and fluorescent click chemistry10,34. In addition to biochemical assays, structural analysis continues to play a vital role in studying PKSs, paving the way for a more detailed understanding of the mechanism and dynamics of PKSs, particularly with the advent of structural knowledge of full-length megasynthases. New strategies have recently been developed to address the technical challenges in obtaining structural information for large PKS protein complexes. For example, small-angle X-ray scattering (SAXS) analyses of megasynthases combined with the rigid-body refinement of the high-resolution domain structures have been used to model modular structures of DEBS, leading to the proposal of a disc-shaped module that can cage the ACP at the center of a ring formed by the other PKS domains35. This method can be used to probe the solution state and obtain structural information for catalytically active megasynthases, and it is a powerful approach for modeling large, dynamic macromolecular complexes. In parallel, electron cryo-microscropy has also recently been used to determine the reconstruction of a full-length PKS module and showed that a single reaction chamber provides the intramodular ACP with access to all of the catalytic sites while the ACP from the upstream module uses a separate entrance to deliver the polyketide intermediate36. An accompanying study further examined this PKS in different catalytic states, providing new insight into the structural rearrangements involved in chain elongation37. Thus, electron cryo-microscopy is emerging as a powerful tool for studying complex PKSs by enabling high-resolution information about the overall structure, organization, and dynamics of complete PKS modules to be obtained directly.\n\nIn addition to in vitro analysis, in vivo study of PKSs, particularly heterologous expression of PKSs and their auxiliary enzymes, has proven to be invaluable for the understanding and production of polyketides, as reflected by the recent developments in new heterologous expression tools and the successes in polyketide production by a wide range of heterologous hosts. While phage-mediated homologous recombination such as λ Red/ET recombineering has long been used for the direct capture of gene clusters from bacterial artificial chromosomes and genomic DNA38, transformation-associated recombination (TAR)-based techniques that typically rely on homologous recombination in yeast and allow for the capture of much larger clusters are quickly gaining traction3,39–42. The development of a Saccharomyces cerevisiae−Escherichia coli shuttle−actinobacterial chromosome integrative capture vector for use with TAR further demonstrates the relative ease and speed with which gene clusters can now be heterologously expressed43. The selection of the heterologous host also remains an important consideration for polyketide production, and popular choices of hosts currently include E. coli, S. cerevisiae, Streptomyces, and Aspergillus, which are all continually being engineered to promote higher compound titers3,39,44. In general, it is best to use a host similar to the native one, as differences in regulation, codon usage, and biosynthetic precursors may result in difficulties with heterologous expression. Nonetheless, the complete refactoring of gene clusters may be used to achieve heterologous expression in hosts that differ significantly from the native producers, and efforts to refactor gene clusters by adding promoters or deleting regulatory elements as well as to engineer the metabolism of heterologous hosts for increased product titers are now easier than ever with tools like CRISPR/Cas, TAR, and λ Red/ET recombineering3,39,43,45–48.\n\n\nConcluding remarks\n\nLooking to the future, an improved understanding of the intriguing PKS machinery remains critical for the successful engineering of polyketide synthesis, and biochemical, in particular quantitative and mechanistic, analyses as well as structural studies of these megasynthases will continue to play important roles in revealing the underlying PKS programming rules. Meanwhile, this research field will continue to benefit from advances in research toolkits, especially new bioinformatics, synthetic biology, and analytical tools, all of which can help lead to the production of new polyketide compounds for drug discovery and development.\n\n\nAbbreviations\n\nPKS, polyketide synthase; CoA, coenzyme A; KS, ketosynthase; AT, acyltransferase; ACP, acyl carrier protein; DEBS, 6-deoxyerythronolide synthase; CCR, crotonyl-CoA carboxylase/reductase; DH, dehydratase; KR, ketoreductase, NR-PKS, non-reducing polyketide synthase; SAT, starter unit ACP transacylase; TE, thioesterase; NRPS, nonribosomal peptide synthetase; SAXS, small angle X-ray scattering; TAR, transformation-associated recombination.", "appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThe authors are supported by the Pew Scholars Program, the National Institutes of Health (DP2AT009148), and the University of California Cancer Research Coordinating Committee.\n\n\nReferences\n\nKhosla C, Herschlag D, Cane DE, et al.: Assembly line polyketide synthases: mechanistic insights and unsolved problems. 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PubMed Abstract \| Publisher Full Text\n\nFeng Z, Kim JH, Brady SF: Fluostatins produced by the heterologous expression of a TAR reassembled environmental DNA derived type II PKS gene cluster. J Am Chem Soc. 2010; 132(34): 11902–3. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nRoss AC, Gulland LE, Dorrestein PC, et al.: Targeted capture and heterologous expression of the Pseudoalteromonas alterochromide gene cluster in Escherichia coli represents a promising natural product exploratory platform. ACS Synth Biol. 2015; 4(4): 414–20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi Y, Li Z, Yamanaka K, et al.: Directed natural product biosynthesis gene cluster capture and expression in the model bacterium Bacillus subtilis. Sci Rep. 2015; 5: 9383. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "12616", "date": "23 Feb 2016", "name": "Gavin 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", "responses": [] }, { "id": "12617", "date": "23 Feb 2016", "name": "Janet Smith", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12618", "date": "23 Feb 2016", "name": "Ikuro Abe", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12619", "date": "23 Feb 2016", "name": "Gong-Li Tang", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-208
https://f1000research.com/articles/5-207/v1	23 Feb 16	{ "type": "Review", "title": "How special is the biochemical function of native proteins?", "authors": [ "Jeffrey Skolnick", "Mu Gao", "Hongyi Zhou", "Mu Gao", "Hongyi Zhou" ], "abstract": "Native proteins perform an amazing variety of biochemical functions, including enzymatic catalysis, and can engage in protein-protein and protein-DNA interactions that are essential for life. A key question is how special are these functional properties of proteins. Are they extremely rare, or are they an intrinsic feature? Comparison to the properties of compact conformations of artificially generated compact protein structures selected for thermodynamic stability but not any type of function, the artificial (ART) protein library, demonstrates that a remarkable number of the properties of native-like proteins are recapitulated. These include the complete set of small molecule ligand-binding pockets and most protein-protein interfaces. ART structures are predicted to be capable of weakly binding metabolites and cover a significant fraction of metabolic pathways, with the most enriched pathways including ancient ones such as glycolysis. Native-like active sites are also found in ART proteins. A small fraction of ART proteins are predicted to have strong protein-protein and protein-DNA interactions. Overall, it appears that biochemical function is an intrinsic feature of proteins which nature has significantly optimized during evolution. These studies raise questions as to the relative roles of specificity and promiscuity in the biochemical function and control of cells that need investigation.", "keywords": [ "Protein structure", "protein interactions", "protein conformation", "biochemical function", "native proteins", "Enzymatic active sites" ], "content": "Introduction\n\nOften proteins adopt a unique, thermodynamically stable native conformation that can perform an amazing variety of biochemical functions ranging from enzyme catalysis and signal transduction to force generation1. When one looks at the diversity of protein functions, one cannot but wonder how they came about. At first glance, the natural tendency is to assume that their remarkable properties mainly arise from evolutionary selection, with the inherent background features that reflect the physical properties of proteins playing a minor role. If so, proteins should exhibit little intrinsic background function, and those that do should be very rare2–8. The fundamental problem with this viewpoint is that for selection to occur, there must be some background function on which to select; in practice, low-level function emerges remarkably quickly in function design studies9–11. The key issue is how to estimate this random background probability for function. Here, computer experiments can provide important insights12–16. For function to occur, often there must be an interaction between molecules. Thus, in what follows, we examine the inherent ability of proteins to engage in small molecule protein-protein and protein-DNA interactions. Surprisingly many biochemical properties of native proteins are found in a library of stable artificial structures generated without any selection for biochemical function. Remarkably, this includes enzymatic active sites and, at much lower frequency, pockets that loosely resemble the enzymatic binding pocket. This suggests that functional selection by evolution is most likely involved in fine-tuning rather than in generation of intrinsic function. If so, marginally stable proteins are inherently ready to engage at low level in the biochemical functions necessary for life.\n\n\nGeneration of an artificial protein library to examine their intrinsic functional features\n\nTo separate out the intrinsic properties of proteins from those due to evolution, one could design proteins without selection for function, solve their structures, assay their function, and explore their similarity to native proteins17–19. To cover all representative protein functions would be a long, expensive process that is, at present, impractical. Rather, we chose to perform a series of computer experiments12–16, where a library of compact homopolypeptides from 40–250 residues in length were generated using the TASSER structure prediction algorithm20. Then, sequences with protein-like composition were selected by optimizing their thermodynamic stability in the putative fold of interest13. These artificial proteins are termed the “ART” protein library.\n\n\nSmall molecule ligand-binding pockets\n\nHaving the ART library in hand, we compared the small molecule ligand-binding pockets to those in native proteins. Remarkably, all ligand-binding pockets in native proteins have a statistically significant match to the pockets in the ART library. This suggests that the library of all ligand-binding pockets, the “pocketome”21, is likely complete and arises from defects in packing of compact secondary structures, as proteins without secondary structure have tiny pockets that cannot bind biologically relevant molecules22. In practice, for single-domain globular proteins, the space of protein pockets is covered by a remarkably small number (about 500) of representative pockets. These results are consistent with a large-scale study on a non-redundant set of ~20,000 known ligand-binding pockets that finds their structural space is crowded, likely complete, and represented by a similar number of pockets23. Similar protein pockets occur in proteins that have globally unrelated folds. On the other hand, closely related proteins need not have similar pockets. The presence of similar pockets capable of binding similar, if not identical, ligands in multiple protein families rationalizes at least part of the reason why drugs have unintended side effects.\n\n\nAbility of ART proteins to bind small molecule metabolites\n\nA representative set of 1400 Kyoto Encyclopedia of Genes and Genomes (KEGG) molecules (clustered using Tanimoto coefficient TC=0.7 from a total 12,271 molecules24) were screened against a representative set of ART proteins using the FINDSITEcomb virtual ligand screening algorithm25. FINDSITEcomb has an average success rate of 21% at identifying micromolar or better binders when 50 or fewer small molecules are screened26. Enrichment factors of the top 1% of ranked ligands relative to a set of 69,271 background molecules (the ZINC 8 library27) culled with a TC28 of 0.7 were 2.57, with 98.6% of ligands having an enrichment factor >1 (the random background result). We found that the median number of binding targets per KEGG molecule is 35, quite close to the number (38) of proteins predicted to bind to drugs in the human exome29. Of these 1400 molecules, 1186 or 84.7% molecules have at least one binding target, and the median number of small molecules that bind per protein is 36 (as compared to 57 drugs per protein, but this discrepancy may be due to the small number of metabolites considered).\n\nWe next explored the enrichment factor of metabolites predicted to bind to proteins in a given metabolic pathway. We define the enrichment factor of a pathway as\n\n\n\nThe average enrichment factor of 238 KEEG pathways is 14.6 with 84.0% of pathways having an Ep >1. Thus, there is a significant tendency for metabolites in existing pathways to bind to ART proteins even without any functional selection. As shown in Table 1, the top 18 most enriched pathways by FINDSITEcomb include ancient pathways associated with glycolysis30, the metabolism of ancient amino acids alanine, aspartate, and glutamate31,32, and glycerolipid metabolism33. Thus, a subset of the top 18 pathways is believed to be ancient. However, the ability to bind a molecule is a necessary but insufficient condition for enzymatic activity, an issue we turn to next.\n\n\nEnzymatic active sites\n\nWe next explored how special the active sites in enzymes are. To address this question, we undertook a large-scale search for amino acids with similar geometry and same residue identity as in enzyme active sites found in a manually curated set from the Catalytic Site Atlas (CSA) database34. There, each entry corresponds to a protein chain with an experimentally determined structure in the Protein Data Bank (PDB)35. In total, we studied 1373 protein chains that are annotated as being enzymes. For each target enzyme, we first detected pockets using a geometry-based method36. We then scanned these pockets against known active sites of the template library of enzymes37. If the target had an amino acid arrangement with a similar geometry as the active sites of a template enzyme whose root-mean-square-deviation (RMSD) from that of the known enzyme’s active site <1 Å RMSD and had 100% sequence identity, we considered it a hit. About 94% of the enzymes hit at least one template enzyme that had different first two-digit Enzyme Commission (EC) numbers, i.e. they are from very different enzyme classes. We further counted hits according to their enzyme classes at the four-digit EC level using various RMSD cutoffs (Figure 1); 75% of target enzymes hit three or more enzyme classes below an RMSD of 1 Å, 54% below a RMSD of 0.75 Å, and 21% below a RMSD of 0.5 Å. Thus, in native proteins, the active sites of enzymes are not as rare nor as geometrically and chemically unique as previously thought; no more than 5000 or so ART structures were searched here.\n\nCumulative fraction of enzymes whose active sites match pocket residues in (A) other classes of enzymes in native structures with different first two digit Enzyme Commission (EC) numbers, (B) in non-enzymes, and (C) in ART structures. For each target enzyme, we count the number of alternative enzyme classes that contain at least a hit by the target enzyme at various root-mean-square-deviation (RMSD) cut-offs.\n\nNext, we performed a search of enzyme-like active sites in native structures of non-enzymes (Figure 1B) and in the ART library (Figure 1C). From a set of 4609 non-enzymes23 and a set of the same number of randomly selected artificial structures, we first identified the largest pocket in these structures, then searched in these pockets for residues that resemble active sites in native enzymes. We only considered hits that had a different global structure with a template modeling (TM)-score <0.438 (a threshold for structural significance) from a target native enzyme. Using the same criteria, at an RMSD <1 Å and 100% coverage and sequence identity, we found at least a hit for 35% of enzyme active sites in non-enzymes and a comparable value (37%) in artificial structures. For an RMSD <0.75 Å, 29% and 31% of native active sites were matched, respectively. Finally, at an RMSD <0.50 Å, 25% and 26% of native active sites were found for non-enzymes and artificial structures, respectively. Small-size active sites were mostly easy to find a hit: about 88% of three-residue active sites, 35% of four-residue active sites, and 0.3% of five-residue active sites were found in artificial structures. About 25% of enzymes had more than four hits in artificial structures. However, it should be pointed out that the global pockets in these matches usually did not have a significant similarity score to the native active site pocket, despite the high structural similarity of their active site residues. Whether these native non-enzymes could weakly catalyze a similar reaction in a different substrate is unknown, as there are other factors that could dictate enzymatic activity39. To further investigate this issue, we froze the catalytic residues in the artificial structure of interest and generated sets of stable sequences for the given fold. We then examined whether artificial pockets globally similar to the active pocket in that native enzyme are generated. As shown in Table 2, depending on the particular ART structure, the success rates (p-values of the pockets <0.05) ranged from 0% to 1.5% of the sequences generated. Given a fixed orientation of the active site residues, there are certain backbone geometries that cannot accommodate the native pocket geometry in certain global folds. Consider, for example, a long narrow pocket. Given the location of the active site residues, it might have to penetrate the backbone for the pocket to be completely recapitulated; clearly, in such a situation, that native enzymatic pocket cannot occur. For successful cases, all of which have a globally unrelated fold to the native structure as assessed by their TM-score40, one need only sample on the order of ~104–105 random sequences to generate a pocket that is at least weakly related to the native pocket. For these, the RMSDs of the aligned residues versus the number of aligned pocket residues for eight pairs of native enzymes-ART proteins are shown in Figure 2. The range of RMSD values is 2–4 Å and spans 4-35 residues. These pockets have p-values <0.05 associated with the pocket similarity (PS)-score37. At this range of PS-scores23, about 13% of ligands share significant chemical similarity as assessed by their TC28.\n\naNo pockets matched even without the active site residue matching restraint imposed.\n\nb8/221,280 pockets match without the active site residue matching restraint imposed.\n\nThe number of residues in the native active site pocket, N, is shown in the figure legend.\n\n\nART protein-protein and protein-DNA complexes\n\nNot only do the ART structures resemble native proteins in terms of fold similarity and ligand-binding pocket but docked ART structures match native protein-protein interfaces, suggesting that the space of protein-protein interfaces is complete and covered by roughly 1000 distinct types of interfaces15. Interestingly, they also possess the ability to form native-like protein-protein and protein-DNA complexes. To demonstrate this, we randomly selected 30,000 pairs of ART structures in representative native-like folds; each fold had 80 protein-like sequences predicted to be stable for that fold. This gives 192 million pairs of ART monomers. To find possible native-like complexes, a simple yet efficient strategy was adopted. First, we compared the backbone structural similarity of ART monomeric structures with monomeric structures found in a library of 1690 non-redundant native dimeric complexes41,42. Using structural alignments, we built putative complexes by superimposing individual ART structures onto their corresponding aligned monomers from the native templates38. We only considered those putative complexes that had significant global structural similarity and were aligned to more than 50% of the native interface. This yielded 135,942 putative ART complexes, and each had a corresponding native protein complex as its template. As shown in Figure 3A, the vast majority were either energetically unfavorable or did not share significant structural similarity to their corresponding template. However, about 2584 ART monomer pairs, or 1.3×10-5 of the total, had strongly favorable interactions and shared significant structural similarity with their templates. These ART complexes may be considered native-like. In general, attractive ART interfaces are enriched in hydrophobic residues. The protein-binding propensity scores of attractive ART complexes overlap with the scores of native complexes (Figure 3B). An example is illustrated in Figure 3C. This ART complex has a favorable interaction energy of -15.443 and shares significant interface similarity (IS) at an IS-score p-value of 7×10-4 with respect to the closest native protein complex42,44. Thus, putative native-like protein-protein complexes are found without any selection whatsoever for protein-protein interactions.\n\n(A) Statistics of putative artificial protein-protein complexes. Joint probability density of interaction energy EPP43 and the p-value of the interface similarity (IS)-score42,44 between an artificial complex and its corresponding native template. Darker blue indicates higher density, with the 100 lowest density spots represented by grey spheres. A vertical/horizontal dashed line is placed at EPP = -15 (a cut-off for high likelihood of interaction) and P = 1×10-3. (B) Protein-binding propensity scores (>0 implies favorable binding) of native protein-protein interfaces versus putatively attractive (EPP <-15) and repulsive (EPP >10) artificial protein-protein interfaces. (C) Example of an ART protein-protein complex. The complex was built by superimposing two artificial structures (cyan and orange) onto a native dimeric template (Protein Data Bank [PDB] code 2f4m, chain A and B, colored in green and purple). Interface alignment according to iAlign42. Both structures are shown in line representations, with the non-interfacial regions of the native template shown in transparent mode for clarity. (D) Statistics of artificial DNA-protein complexes. Joint probability density of DNA-protein interaction energy, EDP46, and the interfacial template modeling (TM)-score22 between an ART protein and its corresponding native template. A vertical/horizontal dashed line is placed at EDP = -10 and iTM-score = 0.4. (E) DNA-binding propensity scores (>0 implies favorable binding) of native DNA-protein interfaces versus putatively attractive (EDP <-10) and repulsive (EDP >10) artificial DNA-protein interfaces. (F) Example of an artificial DNA-protein complex. The complex was built by superimposing the ART structure (red) onto a native template (PDB code 1akh, the native protein and DNA are colored in green and cyan, respectively).\n\nSimilarly, we searched for ART structures with a strong native-like DNA-binding propensity. A set of 32,279 ART folds, each with 80 sequences selected for stability, was scanned. As above, we first performed all-against-all structural comparison between individual ART structures and native protein structures found in 1350 experimentally determined protein/DNA complexes45. The vast majority had either energetically unfavorable DNA-protein interfaces or did not share significant structural similarity with their corresponding native protein templates (Figure 3D). However, 2515 ART proteins, or 9.7×10-4 of the total, had strongly favorable interactions and significant structural similarity to DNA-binding templates. These ART proteins may be considered to have native-like DNA-binding function. Analysis of their DNA-binding interface suggests that they have a large number of positively charged Arg and Lys residues, especially Arg, which is enriched at the DNA-binding interface. This is reasonable, as DNA molecules are negatively charged46. By comparison, DNA-repulsive ART interfaces have a similar sequence composition as native non-DNA-binding surface residues. The DNA-binding propensity scores of DNA-attractive ART structures overlap with the scores of native DNA-binding proteins (Figure 3E); an example is displayed in Figure 3F. Thus, intermolecular interactions between proteins or involving DNA and proteins could emerge without any selection.\n\n\nConclusion\n\nComparison of the properties of native proteins with those of ART structures selected for stability, but not function, shows that many of the properties seen in native proteins emerge as intrinsic features resulting from the packing of secondary structures. The space of small molecule ligand-binding sites found in native and artificial protein structures is shown to be complete, with about 500 representative pockets. Similarly, pockets can occur in proteins with different global folds, while dissimilar pockets are found in proteins that are closely related by evolution with similar structures. Thus, the geometry and amino acid composition of protein pockets are only weakly coupled to the global fold of a protein. The likelihood that a given small molecule differentially interacts with multiple proteins in different families is high. How nature gets around this promiscuity to generate and control cells is a key unanswered question. If cells operated on the basis of one small molecule-one protein target, it is easy to understand how the organized biochemical processes of life occur, but this is apparently not the case29. In practice, the situation is possibly more complex.\n\nRemarkably, ART proteins are predicted to bind weakly to a sufficient number of native metabolites that metabolic pathways are enriched relative to what would be expected at random. Moreover, the ART library has significant matches to the active sites and their associated pockets of enzymes in native proteins (which also are found in putative non-enzyme native proteins). Thus, active site geometry is not special, and it appears that a significant fraction of the biochemistry of life, at least at very low level, is encoded in the physical properties of proteins. If this view is true, and these observations need to be experimentally validated, this has significant implications for the origin of life.\n\nTurning to the likelihood of protein-protein and protein-DNA interactions occurring at random, the strong implication is that a tiny fraction of proteins can engage in at least intermolecular interactions without functional selection. Once again, intermolecular interactions emerge as an inherent feature of proteins due to the packing of secondary structures22. Again, there is the implication of weak omnipresent promiscuous interactions in a cell. How cells sort out the myriad of weak interactions relative to the small fraction of specific ones needs to be better clarified. Part of the answer may lie in subcellular localization.\n\nOverall, these studies suggest that the “special” functional properties of proteins are not as special as commonly viewed. Pockets, enzymatic active sites, and native-like protein-protein and protein-DNA interactions are found in artificial protein structures that are selected for stability and nothing more. The packing of secondary structure is found to provide the geometric context for pockets and intermolecular interfaces. The requirements that a protein be compact and water soluble and adopt a thermodynamically unique conformation give rise to protein sequences that recapitulate the necessary functional features (at least at low level) of real native proteins. Overall, it appears that biochemical function is merely an intrinsic feature of proteins that nature has then significantly optimized.", "appendix": "Competing interests\n\n\n\nThe author(s) declare that they have no competing interests.\n\n\nGrant information\n\nThis research was supported in part by grant no. GM-48835 of the Division of General Medical Sciences of the National Institutes of Health.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nAlberts B, Wilson JH, Hunt T: Molecular biology of the cell. 5th ed. New York, N.Y., Abingdon: Garland Science; 2008. Reference Source\n\nKhersonsky O, Malitsky S, Rogachev I, et al.: Role of chemistry versus substrate binding in recruiting promiscuous enzyme functions. Biochemistry. 2011; 50(13): 2683–90. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nTawfik DS: Messy biology and the origins of evolutionary innovations. Nat Chem Biol. 2010; 6(10): 692–6. PubMed Abstract \| Publisher Full Text\n\nKhersonsky O, Tawfik DS: Enzyme promiscuity: a mechanistic and evolutionary perspective. Annu Rev Biochem. 2010; 79: 471–505. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "12609", "date": "23 Feb 2016", "name": "Vajda Sandor", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12611", "date": "23 Feb 2016", "name": "Ron Elber", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-207
https://f1000research.com/articles/5-206/v1	23 Feb 16	{ "type": "Review", "title": "Management of children with prolonged diarrhea", "authors": [ "Antonietta Giannattasio", "Alfredo Guarino", "Andrea Lo Vecchio", "Antonietta Giannattasio", "Andrea Lo Vecchio" ], "abstract": "Prolonged diarrhea is usually defined as acute-onset diarrhea lasting 7 days or more, but less than 14 days. Its trend has been declining in recent years because of improvement in the management of acute diarrhea, which represents the ideal strategy to prevent prolonged diarrhea. The pathogenesis of prolonged diarrhea is multifactorial and essentially based on persistent mucosal damage due to specific infections or sequential infections with different pathogens, host-related factors including micronutrient and/or vitamin deficiency, undernutrition and immunodeficiency, high mucosal permeability due to previous infectious processes and nutrient deficiency with consequential malabsorption, and microbiota disruption. Infections seem to play a major role in causing prolonged diarrhea in both developing and developed areas. However, single etiologic pathogens have not been identified, and the pattern of agents varies according to settings, host risk factors, and previous use of antibiotics and other drugs. The management of prolonged diarrhea is complex. Because of the wide etiologic spectrum, diagnostic algorithms should take into consideration the age of the patient, clinical and epidemiological factors, and the nutritional status and should always include a search for enteric pathogens. Often, expensive laboratory evaluations are of little benefit in guiding therapy, and an empirical approach may be effective in the majority of cases. The presence or absence of weight loss is crucial for driving the initial management of prolonged diarrhea. If there is no weight loss, generally there is no need for further evaluation. If weight loss is present, empiric anti-infectious therapy or elimination diet may be considered once specific etiologies have been excluded.", "keywords": [ "Prolonged Diarrhea", "Persistent Diarrhea", "Children", "Malnutrition" ], "content": "Introduction and context\n\nDiarrheal disorders are a major health problem in pediatrics worldwide. Accounting for more than 750,000 deaths in children under the age of 5 per year, they are the second leading cause of death in this population according to the World Health Organization (WHO)1. Definitions of diarrheal episodes are usually based on the duration of symptoms rather than etiology. However, there is little consistency and agreement on the definition of acute, prolonged, persistent, and chronic diarrhea in pediatric and adult subjects and in developed and developing countries. In a systematic review on 138 trials, Johnston et al. identified 64 different definitions of diarrhea and 69 definitions of diarrhea resolution. The definitions provided by the WHO were the most commonly used (Table 1)2.\n\nMost diarrheal illnesses last 5–7 days and are due to self-limiting intestinal infections. These episodes are usually defined as acute diarrhea (AD). According to WHO, episodes of diarrhea are usually classified as AD when they last up to a maximum of 14 days and “persistent or chronic diarrhea” when they last >14 days3. However, a subset of children experiences an acute-onset diarrhea lasting 7 days or more, but less than 14 days; this may be defined as prolonged diarrhea (ProD) and usually indicates an episode that continues beyond the expected duration of a typical acute infectious diarrhea4. This entity accounts for a relatively small but consistent number of diarrheal episodes that, especially in developing countries, is associated with a high risk of mortality and morbidity4. In this review, we define ProD as an episode of diarrhea of acute onset lasting 7 to 13 days and persistent diarrhea (PD) as diarrhea lasting 14 or more days.\n\n\nEpidemiology and risk factors\n\nData on the etiology and epidemiology of ProD are limited because of the huge variability in defining diarrheal disorders. Some evidence may be extrapolated from studies reporting data on PD5–7.\n\nIn a study in Israeli children, about a quarter of the study population presented with ProD (lasting 8–13 days) and 14–18% with PD (14 or more days’ duration)6. However, Moore et al. firstly proposed the definition of ProD as a specific disorder and reported an incidence of 12% of all diarrheal cases in a large Brazilian cohort, accounting for a quarter of all days of diarrhea recorded in the 10-year study period4. In the same population, less than 5% presented with PD. It should be noted that when a diarrheal episode progresses from acute to ProD, there is a 6-fold higher risk that the episode will evolve into PD4.\n\nProD is more common in children aged 6 to 24 months and peaks in the second semester of life4. Children who developed ProD in their first year of life have a doubled risk of developing PD at pre-school age4. In addition, children experiencing severe diarrhea and dysenteric illnesses with blood and mucus in their stools are more likely to present with a course longer than those who present with mild-to-moderate diseases8,9.\n\nThese findings, that demonstrate a close relationship between ProD and PD, may be due to different mechanisms: on one hand, ProD affects child growth and mucosal immunity and impacts on gut microflora and intestinal barrier functions; on the other hand, the increased risk of subsequent episodes may be related to specific individual features or to genetic, nutritional, or environmental characteristics that predispose to persistent intestinal illnesses. Mainly in developing areas, ProD is linked with malnutrition in a complex cause-effect relationship, implicating a multifactorial “vicious cycle” involving intestinal infections, microflora disruption, micronutrient deficiency, and immunodeficiency. The role of malnutrition is supported by the evidence that non-breast-fed children and those who are weaned early or recently exposed to formula, as well as children with underlying malnutrition, vitamin deficiency, and wasting, are at increased risk of developing ProD4,8–10.\n\nEnvironmental factors also contribute to ProD, since living in poor areas with poor hygiene conditions and low mothers’ education expose children to a doubled risk of developing ProD4.\n\nFinally, the risk of ProD is reduced by half for 10 years’ increase in maternal age, and if a mother completes primary school, the risk of ProD and PD in her child decreases6.\n\nIrrespective of the etiology and risk factors, children with ProD have a higher risk of nutritional derangement, micronutrient deficiency, risk of developing PD, infections, and immunodeficiency.\n\n\nEtiology and pathophysiology\n\nThe pathogenesis of ProD is multifactorial and essentially based on 1) persistent mucosal damage due to specific agents or sequential infections with different pathogens, 2) host-related factors including micronutrient and/or vitamin deficiency, undernutrition, and immunodeficiency, 3) high mucosal permeability due to previous infectious processes and nutrient deficiency with consequent malabsorption, and 4) microbiota disruption (Figure 1). In some cases, ProD may represent the onset of chronic intestinal disorders including celiac disease, inflammatory bowel disease (IBD), and autoimmune enteropathies that are usually characterized by PD.\n\n“Vicious cycle” of prolonged diarrhea involves intestinal infections, microflora disruption, micronutrient deficit, undernutrition, and immunodeficiency.\n\nHIV: human immunodeficiency virus.\n\nIn children with PD, electron microscopy shows shortening of villi, decrease in number and height of microvilli, blunting of borders of enterocytes, loss of glycocalyx, and presence of mucous pseudomembranes coating the epithelial surface. In addition, children with PD often have marked mucosal inflammation when compared to children with AD, presenting higher interferon-gamma response, significant elevation of fecal lactoferrin, interleukin (IL)-8, and IL-1β, and a higher percentage of CD8+ T cells11–13. More than half of children with severe AD or ProD develop protein-losing enteropathy14. Also, children with ProD share some of these immunological characteristics.\n\nAlthough infections are a major cause of ProD, there is no clear evidence of a role for selected pathogens in inducing ProD6,8,14.\n\nIn developing countries, many of the bacterial pathogens responsible for AD and dysentery also cause PD. Invasive diarrhea due to Shigella contributes to ProD and PD, and the prevalence of Shigella among children with ProD and PD is higher than in those with AD6,8,15–17. Enteropathogenic Escherichia coli have been found in at least 25% of children tested in low- and middle-income countries and may cause ProD in some settings5,7,8.\n\nSelected viruses may be responsible for ProD. Rotavirus, norovirus, and sapovirus have been found in as many as 50% of diarrheal episodes resolving within the third week in children living in the United States18. In some cases, such as rotavirus infection, ProD could be due to susceptibility to other infections or to nutrient malabsorption rather than being the direct result of the pathogenic agent11. About 10% of astrovirus infections may complicate AD with prolonged episodes of diarrhea, even in non-immunocompromised patients19.\n\nParasites including Giardia, Cryptosporidium, and Cyclospora have been related to ProD and PD in developing areas. Cryptosporidium, a common agent of ProD and PD in HIV-infected children, was frequently isolated also in immunocompetent children4.\n\nAlthough 35–70% of children with PD tested positive for at least one pathogen, multiple isolations are common, making it difficult to distinguish bystanders from agents causing illness5,6.\n\nIn addition, the pattern of microorganisms that tends to be associated with ProD is different in developed and developing countries. In the former, viruses are more frequently found, whereas in developing countries, specific bacteria and protozoa are more common.\n\nA further mechanism that may cause ProD is so-called small intestinal bacterial overgrowth (SIBO). It involves the colonization of the small intestine by bacteria that are usually found in the colonic microbiota or an increase in their number. SIBO is more common in children with underlying intestinal diseases, such as blind loop syndrome, dysmotility, or inflammatory diseases of the intestine. However, SIBO may be a possible complication of AD due to recent infection, the presence in the intestinal lumen of carbohydrates and short-chain fatty acids, the temporary alteration of gut motility, or previous antimicrobial use. It has been associated with antacid therapy. The bacterial overgrowth in the small intestine causes inflammation and malabsorption of liposoluble vitamins and steatorrhea with further worsening of diarrhea and malnutrition.\n\nProD and PD are commonly seen in association with malnutrition and micronutrient deficiencies in developing areas. The latter conditions cause the impairment of immunological mechanisms for clearing infections as well as delayed intestinal repair, thereby contributing to the vicious cycle between diarrhea and malnutrition20.\n\nPD is also due to malabsorption of select nutrients. Food intolerance and food allergy may be responsible for ProD. The mucosal damage secondary to infection leads to loss of lactase activities with a consequent malabsorption of carbohydrates that, due to their osmotic power, worsen and perpetuate diarrhea.\n\nIn addition, the increased permeability related to both acute infection and underlying malnutrition may potentially lead to food-antigen sensitization.\n\nSome children and adolescents may develop functional gastrointestinal symptoms, including PD, following a single infectious episode of diarrhea. The clinical picture is characterized by diarrhea and abdominal pain without loss of body weight. This is a syndrome defined as chronic non-specific diarrhea of childhood or “toddler’s diarrhea” and is currently named post-infectious irritable bowel syndrome. The Rome III criteria define the type of symptoms and their duration in age groups21. Based on age-related criteria, the diagnosis can be made without further investigation.\n\n\nDiagnostic approach\n\nThe management of ProD is complex because the etiology and pathogenesis are complex. Furthermore, the majority of available data on management and treatment are focused on PD rather than on ProD and very few studies have examined ProD as a distinct category4.\n\nIn developing countries, the main consequences of ProD are nutritional derangement, morbidity (with an increased risk of hospital admission), and even death14. In this setting, it is difficult to perform expensive and time-consuming tests to identify the etiology of ProD. Optimal management of AD is the ideal strategy to prevent ProD. It includes appropriate fluid replacement, zinc treatment, and optimal nutrition in developing countries22. Rotavirus vaccination, promotion of breast-feeding, promotion of hand washing, improved water supply and quality, and community-wide sanitation indirectly prevent ProD23,24. However, the diagnostic approach to and the treatment of ProD is a challenge. Because of the wide etiologic spectrum, diagnostic algorithms of ProD should also include the age of the patient, clinical and epidemiological factors, and the results of microbiological investigations, when available. An algorithm for ProD is provided in Figure 2. The algorithm is intended as a general tool, and decisions must take into consideration medical history and clinical features of individual cases as described below.\n\nSpecific clues in the family and personal history (previous episodes of AD or ProD, history of chronic diseases or food allergy, and immunological status) may provide useful indications.\n\nInitial clinical examination should include the evaluation of general and nutritional status and the presence of weight loss. Dehydration requires prompt supportive interventions to stabilize the child. Malnutrition may precede the onset of diarrhea, contributing to its duration, or it could be the consequence of the disease. The presence or absence of weight loss may help drive the subsequent diagnostic and therapeutic approach (Figure 2).\n\nSome children may lose weight as a consequence of poor caloric input after an episode of AD, and in these cases, reintroduction of a free diet is generally effective. However, most children with ProD and weight loss deserve specific medical interventions and careful follow up. If first-line therapeutic interventions do not result in a clear clinical improvement and diarrhea persists, a referral to the gastroenterologist is needed (Figure 2).\n\nIf there is no evidence of weight loss, in most cases there is no need for further investigation. However, a clinical re-evaluation with assessment of the trend of symptoms and weight and revision of nutritional regimens may be considered. Children with persistence of symptoms may need to be referred to the gastroenterologist for further care.\n\n*Feeding pattern should be normalized according to the “4F” role: fat (increase dietary lipids to at least 35–40% of total daily energy intake), fiber (normalize fiber intake by introduction of fruits and wholegrain bread), fluid (restrict fluid intake if history is significant for high fluid consumption), and fruit juice (discourage overconsumption of fruit juices, especially those containing sorbitol or a high fructose/glucose ratio).\n\n§ Empiric antibiotic treatment should cover most probable enteric infections (Shigella and enteropathogenic Escherichia coli) and/or small intestine bacterial overgrowth.\n\nThe initial diagnostic workup for children with ProD should include stool cultures and a search for parasites and enteric viruses. However, the wide pattern of microorganisms potentially involved requires high-level techniques.\n\nAlthough traditional culture is still an invaluable tool in clinical settings, in some instances other techniques are needed for the identification and differentiation of bacterial species25. For other pathogens, it is more important to identify the toxins than the organisms themselves, as in the case of enterotoxigenic E. coli, Shiga toxin of enterohemorrhagic E. coli, and Clostridium difficile26.\n\nLight microscopy represents the traditional technique used to diagnose intestinal parasites. However, its sensitivity depends on the burden of infection, the stage and delivery to the labs of the specimen, and the experience of the observer26. More sensitive and specific enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) analysis are used to detect protozoa in fecal samples, but these assays are still not routinely available.\n\nEnteric viruses are commonly searched for by means of ELISA and latex agglutination analysis. However, molecular genetic techniques detect a wider spectrum of pathogens than conventional techniques and provide information about their molecular epidemiology25. Again, these techniques are not readily available in primary care settings and in resource-limited areas.\n\nA search for SIBO should also be included in the diagnostic work-up of ProD. Breath hydrogen test may identify an abnormal bacterial proliferation in the small bowel; it can also be used to detect carbohydrate malabsorption27. However, the sensitivity and specificity of this test is low and it is difficult to perform, mainly in young children. Alternatively, a simple measurement of stool pH or a test for reducing substances (Clinitest) may be easily done at the bedside to investigate carbohydrate (usually lactose) malabsorption. Other specific diagnostic tests, including evaluation of intestinal function and inflammation, imaging, and endoscopy, should be performed in case of persistence of diarrhea in the setting of pediatric gastroenterology.\n\nNon-invasive diagnostic tests for intestinal and pancreatic function (including dual absorption test, xylosemia, iron oral load, lipase, and fecal elastase) and inflammation (fecal calprotectin and last ileal loop abdominal ultrasound) may provide useful information for diagnosis, when available27,28. Although abdominal ultrasound may be affected by subjective evaluation if an expert consultant is available, it may be of support in management. The association of a negative scan of last ileal loop with negative fecal calprotectin significantly reduces the possibility of IBD28. The use of noninvasive diagnostic tests in the diagnostic algorithm may reduce invasive procedures. However, if diagnosis is not obtained otherwise, endoscopy with biopsy should be considered.\n\nIn young children with a familial history of atopy and suggestive personal history and clinical features, skin tests for the screening of food allergy (including patch tests and prick tests) may be performed and elimination diet with subsequent challenge should be considered29.\n\n\nTreatment\n\nExpensive laboratory evaluations usually are of little benefit in guiding the successful treatment of ProD, and an empirical therapeutic approach may be effective in the majority of cases (Figure 2).\n\nIf there is no weight loss, the approach should be conservative and no investigations are generally needed. A review of the diet is worthwhile, including a check on a possible excess of sugar-containing drinks as a cause of diarrhea. Feeding pattern should be normalized according to the “4F” rule: fat (increase dietary lipids to at least 35–40% of total daily energy intake), fiber (normalize fiber intake by introduction of fruits and wholegrain bread), fluid (restrict fluid intake if history is significant for high fluid consumption), and fruit juice (discourage overconsumption of fruit juices, especially those containing sorbitol or with a high fructose/glucose ratio).\n\nA lactose-free diet can be started if weight loss is present. Some children with ProD who do not have weight loss at first presentation may benefit from a lactose-free diet since disaccharidase deficiency secondary to AD is relatively common. Exclusion diets are usually administered with the double purpose of overcoming food intolerance, which may be the primary cause of ProD, or its complication. The sequence of elimination should be graded from less (e.g. cow’s milk protein hydrolysate) to more restricted diets (amino acid-based formula) according to the child’s clinical conditions. This approach should be reserved for infants and young children.\n\nProD is often an infection-induced illness in the majority of cases30. However, considering that pathogens associated with ProD are also often found in healthy children without diarrhea7, even when an enteric pathogen is detected, it is not always clear that this is the cause of the illness.\n\nAntimicrobial agents are indicated for the treatment of selected parasites31 and selected enteropathogenic bacteria, such as enteropathogenic E. coli and enteroaggregative E. coli14. Pathogens such as Shigella and Cryptosporidium are commonly associated with ProD in tropical, developing countries and should be treated in case of ProD4.\n\nNitazoxanide is a broad-spectrum antimicrobial agent with activity against protozoa, nematodes, cestodes, trematodes, and bacteria, with a favorable safety profile32,33. It is effective in childhood cryptosporidiosis34 but not consistently in undernourished children or in HIV-infected patients35. Anecdotal cases of children successfully treated with nitazoxanide because of PD (<30 days) have been reported36. This strategy seems to be effective in select situations, saving time-consuming tests to identify the cause of diarrhea36.\n\nMetronidazole can be used for Giardia, and trimethoprim-sulfamethoxazole can be used for Cyclospora and as a second-line antibacterial drug for a number of pathogens14.\n\nVery few data on the treatment of viral ProD are available. Human immunoglobulin, available for intravenous use, may be administered orally (300 mg/kg of body weight) in a single dose. The rationale of passive immunotherapy is based on the demonstration of neutralizing antibodies against all viruses in a medical preparation of immunoglobulins37. They are found in the stools after administration, and this treatment reduces the duration of stay in severe and/or immunocompromised patients with AD and in patients with severe diarrheal episodes due to rotavirus38,39. This treatment seems to also have a potential role in immunocompromised patients with norovirus enteritis40. In this population, a positive trend towards resolution of diarrhea and decreased stool output in the treatment group compared with placebo was found, but no benefit was reported for length of hospital stay or hospital cost40. However, no data on oral immunoglobulins and viral-ProD is available, with the exception of rotavirus41.\n\nCiprofloxacin is effective in cases of diarrhea associated with enteroaggregative E. coli in HIV-infected adults42. However, its efficacy needs to be proven in a large-scale trial in children with ProD.\n\nThe efficacy of antimicrobials in children with ProD in whom etiology is unknown is even more controversial. In a recent systematic review in young children with PD of unknown or non-specific cause from developing countries, no difference was demonstrated for oral gentamicin or metronidazole compared with placebo, whereas sulfamethoxazole-trimethoprim was more effective than placebo for diarrhea at 7 days and for total stool volume43. To date, the evidence to recommend the use of antibiotics in ProD of unknown or non-specific causes is still limited.\n\nSome data support the use of probiotics in ProD. Lactobacillus spp. and Saccharomyces boulardii significantly reduced the number of stools and duration of diarrhea in children with PD44.\n\nA recent Cochrane review showed that probiotics shortened the duration of diarrhea and reduced stool frequency and hospital stay. However, only four trials with a small number of participants were available for meta-analysis. The authors concluded that, although probiotics appear to hold promise as adjunctive therapy, there is insufficient evidence to recommend their routine use in children with ProD45.\n\nDietary treatment is crucial in ProD. However, nutritional interventions are often expensive and poorly applicable in developing countries. Locally available, inexpensive foods and vitamin and mineral supplementation have been proposed46–48. Locally tailored nutritional interventions are ideal in the developing world: they are inexpensive and culturally acceptable, provide sufficient levels of energy and nutrients to malnourished children, and allow the continuation of dietary therapy at home46,49.\n\nA multicenter study in severely ill children aged 4–36 months with PD showed a high success rate of a dietary regimen using inexpensive, locally available foods (variable association of rice, maize, lentils, chicken, yoghurt, milk, sucrose or glucose, and oil) and vitamin and mineral supplementation46. Although a recent systematic review underlined the low-quality evidence of these studies, the authors found no evidence to support the use of proprietary formulas or specialized ingredients over the use of locally produced and readily available foods in the treatment of either AD or PD50.\n\nOther approaches that have been evaluated include the use of amylase-rich flours in cereal-based porridges to decrease viscosity and thus increase nutrient density and children’s nutrient intake. However, these trials have been performed only in children with AD51. Also, mixed diets including specific ingredients thought or known to have antidiarrheal properties, such as green banana, have been tested. These reports showed a significantly higher cumulative probability of recovery in children52,53.\n\nChildren with ProD and malnutrition may present with deficiency of selected micronutrients such as vitamin A, zinc, folic acid, copper, and selenium54. Zinc was found to have therapeutic efficacy (typically resolution of small bowel damage and shortening duration of diarrhea) in several trials of PD55. Zinc and vitamin A in combination seem to be even more effective than either vitamin A or zinc alone in reducing PD in developing countries56.\n\n\nConclusion\n\nIn children with severe malnutrition, ProD may be the direct result of secondary immunodeficiency and the consequence of a reduction of intestinal absorptive-digestive surface. In this condition, nutritional interventions (nutritional regimen review/optimization and micronutrient supplementation) should be considered with the aim of optimizing the use of residual functioning intestine over time. Enteral nutrition given continuously by feeding tube may be used in severe cases of reduction of intestinal functional surface. However, anti-infectious therapy should also be considered.\n\nOverall, the treatment of ProD is a balance between a possible infectious etiology and a nutritional approach. When, however, the features of irritable bowel syndrome (defined according to the Rome III criteria) are met, no specific medication should be prescribed. However, parental reassurance and diet tailoring (e.g. an increase in fiber intake) may be helpful, and counseling/behavioral therapy may be indicated to reduce anxiety that may trigger gastrointestinal symptoms. If a specific cause of diarrhea is detected, such as celiac disease or IBD, an appropriate therapy should be started.\n\n\nAbbreviations\n\nWHO, World Health Organization; AD, acute diarrhea; ProD, prolonged diarrhea; PD, persistent diarrhea; IBD, inflammatory bowel disease; SIBO, small intestinal bacterial overgrowth.", "appendix": "Author contributions\n\n\n\nAGi, AG, and ALV devised the paper. AGi and ALV carried out the systematic literature research. AGi and ALV prepared the first draft. AG provided substantial contribution to the preparation of the manuscript. ALV and AGi prepared figures and tables. 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\nReferences\n\nWorld Health Organization: Diarrhoeal disease. 2013. Reference Source\n\nJohnston BC, Shamseer L, da Costa BR, et al.: Measurement issues in trials of pediatric acute diarrheal diseases: a systematic review. Pediatrics. 2010; 126(1): e222–31. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization: Persistent diarrhoea in children in developing countries: memorandum from a WHO meeting. Bull World Health Organ. 1988; 66(6): 709–17. 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PubMed Abstract \| Free Full Text \| Faculty Opinions Recommendation\n\nMitra AK, Rahman MM, Mahalanabis D, et al.: Evaluation of an energy-dense meal liquefied with amylase of germinated wheat in children with acute watery diarrhoea: a randomized controlled clinical trial. Nutr Res. 1995; 15(7): 939–51. Publisher Full Text\n\nRabbani GH, Larson CP, Islam R, et al.: Green banana-supplemented diet in the home management of acute and prolonged diarrhoea in children: a community-based trial in rural Bangladesh. Trop Med Int Health. 2010; 15(10): 1132–9. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nRabbani GH, Teka T, Zaman B, et al.: Clinical studies in persistent diarrhea: dietary management with green banana or pectin in Bangladeshi children. Gastroenterology. 2001; 121(3): 554–60. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nBhan MK, Bhandari N, Bahl R: Management of the severely malnourished child: perspective from developing countries. BMJ. 2003; 326(7381): 146–51. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlack RE: Zinc deficiency, infectious disease and mortality in the developing world. J Nutr. 2003; 133(5 Suppl 1): 1485S–9S. PubMed Abstract \| Faculty Opinions Recommendation\n\nRahman MM, Vermund SH, Wahed MA, et al.: Simultaneous zinc and vitamin A supplementation in Bangladeshi children: randomised double blind controlled trial. BMJ. 2001; 323(7308): 314–8. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation" }	[ { "id": "12606", "date": "23 Feb 2016", "name": "Sean Moore", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12607", "date": "23 Feb 2016", "name": "Hans Hoekstra", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12608", "date": "23 Feb 2016", "name": "Jorge Amil Dias", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-206
https://f1000research.com/articles/5-205/v1	22 Feb 16	{ "type": "Review", "title": "Axonal maintenance, glia, exosomes, and heat shock proteins", "authors": [ "Michael Tytell", "Raymond J. Lasek", "Harold Gainer", "Raymond J. Lasek", "Harold Gainer" ], "abstract": "Of all cellular specializations, the axon is especially distinctive because it is a narrow cylinder of specialized cytoplasm called axoplasm with a length that may be orders of magnitude greater than the diameter of the cell body from which it originates. Thus, the volume of axoplasm can be much greater than the cytoplasm in the cell body. This fact raises a logistical problem with regard to axonal maintenance. Many of the components of axoplasm, such as soluble proteins and cytoskeleton, are slowly transported, taking weeks to months to travel the length of axons longer than a few millimeters after being synthesized in the cell body. Furthermore, this slow rate of supply suggests that the axon itself might not have the capacity to respond fast enough to compensate for damage to transported macromolecules. Such damage is likely in view of the mechanical fragility of an axon, especially those innervating the limbs, as rapid limb motion with high impact, like running, subjects the axons in the limbs to considerable mechanical force. Some researchers have suggested that local, intra-axonal protein synthesis is the answer to this problem. However, the translational state of axonal RNAs remains controversial. We suggest that glial cells, which envelop all axons, whether myelinated or not, are the local sources of replacement and repair macromolecules for long axons. The plausibility of this hypothesis is reinforced by reviewing several decades of work on glia-axon macromolecular transfer, together with recent investigations of exosomes and other extracellular vesicles, as vehicles for the transmission of membrane and cytoplasmic components from one cell to another.", "keywords": [ "Axonal maintenance", "glia", "exosomes", "heat shock proteins", "HSP", "axoplasm" ], "content": "Introduction\n\nThe axon is a unique cellular structure. It extends from the neuron’s cell body as a cylindrical process with a constant, stable diameter and length, differing from all other cellular extensions (including dendrites) that are typically tapered and dynamic in shape1,2. In addition, axons extend for very long distances, often many orders of magnitude greater than the cell body’s diameter and volume. Maintenance of this stable structure is critical for the principal function of the axon, i.e. to conduct the action potential to its synaptic terminal with a determined conduction velocity. How this seemingly fragile cylinder is maintained for the lifespan of an organism, especially in large ones, has puzzled researchers for decades. We undertook this review to integrate historical and contemporary research findings that suggest a mechanism for the maintenance of macromolecules in the mature axon that are critical for its structure and function.\n\nThe traditional view is that the macromolecules (e.g. proteins) that are important for axonal function are synthesized in the neuronal cell body and supplied to the axon by axonal transport mechanisms2–7. The adequacy of this mechanism to compensate for the turnover of proteins in the axon has been challenged8, and it has been posited by several groups that “local” protein synthesis in the axon itself may be needed to supplement the source from the neuronal soma8–11. However, we propose here that a more likely alternative supplementary mechanism is the transfer of macromolecules from the adaxonal glia to the axon by extracellular vesicles (EVs), e.g. exosomes. The idea of glia supplying proteins to vertebrate axons was first proposed by Marcus Singer12–14, who showed by insightful interpretation of autoradiographic results that newly synthesized Schwann cell proteins that had incorporated radioactive amino acids were transferred into the peripheral axons that they ensheathed.\n\nOur thesis here is that glia to neuron transfer of macromolecules is likely to occur via EVs, such as exosomes. We summarize recent advances in exosome research that indicate that exosomes are the primary vehicles for intercellular macromolecular transfer. Additionally, we describe evidence for a unifying principle that a key function of glia-neuron transfer is neuroprotection by heat shock protein (Hsp)-containing exosomes.\n\n\nLessons from invertebrate axons\n\nGiant axons in invertebrates offer a unique opportunity to study the possibility of de novo protein synthesis in axoplasm because pure axoplasm can be collected for physiological and biochemical analyses. The first biochemical experiments that used the squid giant axon isolated from its cell bodies to determine whether axonal protein synthesis occurred were performed by Giuditta and colleagues15. These authors observed the appearance of radioactively labeled proteins in extruded axoplasm and noted that there could be two potential sources of the newly synthesized axonal proteins, intra-axonal protein synthesis or the surrounding glia called Schwann cells. The next important investigations showed that isolated axoplasm from squid and marine worm (Myxicola) giant axons contained transfer RNA, but not detectable ribosomal RNA (rRNA)16, and thus seemed to discount the possibility of de novo protein synthesis in the axoplasm. In this regard, it should be noted that Giuditta and colleagues continue to report finding various components of protein translational machinery in isolated squid axoplasm9,17–21, including rRNA22 and ribosomes23, as support for the concept that de novo protein synthesis could be occurring in the axoplasmic compartment24.\n\nAn alternative proposal about the source of radiolabeled proteins in squid giant axon axoplasm was presented as the “Glia-Neuron Protein Transfer Hypothesis” published in 1977. These publications posited that the source of the newly synthesized radiolabeled proteins found in the axoplasm was the adaxonal Schwann cell sheath25,26. This hypothesis was based on the facts that (1) the giant axon did not contain a significant amount of ribosomes or rRNA; (2) isolated axoplasm was unable to synthesize labeled proteins from radioactive amino acids; (3) injection of RNase into the giant axon did not reduce the appearance of newly synthesized proteins in the axoplasm of the isolated giant axon26; and (4) incubation of the squid giant axon in 3H-leucine while it was cannulated and perfused with an artificial axoplasm solution demonstrated the appearance of newly synthesized proteins in the perfusate, continuously for over 8 hours of incubation, whether or not RNase was included in the perfusion solution. However, when puromycin was included in the extra-axonal incubation solution to block de novo protein synthesis in the glial sheath, it completely prevented the appearance of labeled proteins in the perfusate25. Taken together, these data strongly indicate that the squid giant axon cannot synthesize proteins de novo and that the most likely source of the newly synthesized proteins is its adaxonal Schwann cell sheath.\n\nLater studies by Lasek and Tytell in the squid giant axon strengthened and extended the “Glia-Neuron Protein Transfer Hypothesis”, and focused on the nature of the transported proteins and the possible mechanisms of the intercellular transfer. In one study, the labeled proteins in the axoplasm, the glial sheath containing the Schwann cells, and the stellate ganglion containing the cell bodies of the giant axon were analyzed separately and compared27. Over 80 glial polypeptides were found to be selectively transferred into the axoplasm and many of these were distinct from groups of stellate ganglion proteins, which were presumed to include those destined for the axon via axonal transport. Three of the more highly labeled transferred glial polypeptides were actin, a fodrin-like polypeptide, and a 70–80 kDa polypeptide they named traversin. Traversin was later identified as being an Hsp, since its expression and transfer was increased by exposure of the axon to elevated temperatures, as was another prominent 95 kDa protein28. Since both the 70 kDa and 95 kDa proteins were similar in molecular weight and charge to Hsps described in other systems, this suggested that they were members of the Hsp family and that the glia provided the axon with proteins that may be involved in the reaction to metabolic stress. A follow-up study used a fluorescent vesicular reporter, acridine orange, that selectively stained acidic vesicular structures in glial cells in the giant axon sheath. The results suggested that some of those fluorescent vesicles in the glia were transferred into the axoplasm29. Several speculations for the mechanisms that could produce these transfers were considered. These were glial exocytosis coupled with neuronal endocytosis, diffusion through intercellular channels, modified phagocytosis, and protein translocation analogous to the transfer of newly translated proteins into the rough endoplasmic reticulum (RER)30,31.\n\nIn 1967, Hoy, Bittner, and Kennedy32 made a remarkable observation during studies on axonal regeneration in the crayfish claw. These authors found that after transection of the motor nerves innervating the muscles in the crayfish claw, stimulation of the distal stumps of these nerves could evoke normal synaptic potentials in the muscle more than 100 days after transection32. This functional survival of the crayfish motor axon after transection suggested that the adaxonal glia might contribute to its long-term survival.\n\nSubsequent experiments by Sarne et al.33,34 found that in the transected crayfish nerve the small sensory (cholinergic) axons degenerated, whereas the non-cholinergic motor axons did not. The motor axons were associated with a robust glial sheath, whereas the sensory axons had relatively poor glial coverage, often with many axons sharing a single glial sheath. Incubation of the isolated nerve with radioactive amino acids produced an autoradiographic picture similar to that seen in the squid giant axon experiments, and transection of the axon seemed to produce an increase in the amount of protein synthesized33. Since the neurotransmitters in the sensory axons (acetylcholine) and in the inhibitory motor axons (γ-aminobutyric acid, GABA) were known, Sarne et al.34 studied the effects of transection of these axons from their cell bodies on the contents of their respective synthetic enzymes, choline acetyltransferase (CAT) in the sensory axons and glutamic acid decarboxylase (GAD) in the motor neurons. Transection of the axons in vivo produced a dramatic decrease in CAT activity, presumably due to the degeneration of the cholinergic axons (see also 35), but the GAD activity remained unchanged even 14 days after transection. Complete isolation of the axons in organ culture also did not alter the GAD activity in the tissue. However, incubation of the cultured tissue in the eukaryotic protein synthesis inhibitor anisomycin caused a complete loss of GAD activity. Therefore, it appeared that a local synthesis in the nerve fibers (presumably in the glia surrounding the inhibitory motor axon) was responsible for the maintenance of the GAD activity.\n\nSimilar findings were made by Meyer and Bittner36,37 on the medial giant axon (MGA) in the central nervous system of the crayfish, which showed that the kinetic appearance of the autoradiographic grains representing newly synthesized proteins, first in the glia and later in the axon, were consistent with a transfer of glial proteins to the axon. Bittner and colleagues have done extensive work since then studying the long-term survival of axons in the absence of cell bodies in the central nervous system of crayfish. The survival of the MGA in the crayfish nerve cord was found to be particularly dependent on intercellular transfer of macromolecules from surrounding glia35,38. Of particular interest here is the identification of Hsps of the 70 kDa family as prominent proteins found in the crayfish MGA39. The Hsp70 proteins represented 1–3% of the total protein in the axoplasm of the MGAs. After heat-shock treatment, overall protein synthesis in the glial sheath was decreased compared with that of control axons, but newly synthesized proteins of 72 kDa, 84 kDa, and 87 kDa appeared in both the axoplasm and the sheath and were interpreted as being the stress-inducible Hsps. These authors proposed that the Hsps in the MGAs may help these axons maintain essential structures and functions following acute heat shock.\n\n\nThe significance of intra-axonal RNAs\n\nThe above work clearly showed that glia-axon protein transfer occurs in many but not all axons and is particularly important for the maintenance of large invertebrate axons. This is also true for many mature vertebrate axons12,40. However, there have been recent reports of the presence of intra-axonal RNAs shown to be derived from either axonal transport or glia-axon transfer mechanisms10,41–43. This has led many investigators to infer that the presence of intra-axonal RNAs represents evidence for intra-axonal protein synthesis10,41,42,44. There is also excellent evidence presented that de novo protein synthesis can occur in neuronal compartments other than the cell body, e.g. in dendrites45–48, in growth cones during development and regeneration, and in immature axons in culture10,21,42,44,49. Nevertheless, the presence of mRNAs and other translational machinery in mature axons should not be taken as evidence of de novo protein synthesis in that structure. For example, squid giant axoplasm contains considerable neurofilament protein mRNAs50 but does not synthesize neurofilament proteins (see 26 and Gainer [in preparation]). Another example that the presence of mRNAs does not imply protein synthesis comes from studies of posterior pituitary axons and terminals. Even though these structures contain oxytocin and vasopressin precursor protein mRNAs transported from the hypothalamic magnocellular neurons and they increase in amount with functional activity51–58, they are not used to synthesize the precursor proteins as would occur on the RER in the neuronal cell bodies, probably because RER is absent from the pituitary axons and nerve endings59–61. The issue of whether the presence of intra-axonal RNAs is concordant with evidence of intra-axonal protein synthesis will depend on the development of new methods to visualize protein synthesis in situ62,63. Whether or not local protein synthesis occurs in the axon, in principle, the glia can provide additional proteins not synthesized by the neuron, such as Hsp70, that can protect axonal function in the face of physical trauma and metabolic stress.\n\n\nGlia-axon macromolecular transfer, extracellular vesicles, and Hsp70\n\nIn the 1980s, before exosomes and other types of EVs were recognized as vehicles for the movement of cytoplasmic constituents from one cell to another, it was common knowledge that certain cell types release material by the blebbing of small, membrane-bound packets from their apical surfaces, a process known as apocrine secretion first described in the middle 1800s by several biologists including Purkinje. Mammary gland secretory epithelium and apocrine glands in the skin are two examples of tissues that exhibit this type of secretion. Thus, this concept was familiar when a few reports began to appear in the 1980s that showed other cell types release cytoplasmic proteins during normal function. A now well-known example is in the development of reticulocytes into mature red blood cells (RBCs), which dispose of many of the intracellular and membrane components unneeded in mature RBCs by sequestering things like transferrin receptors and a variety of other cytoplasmic proteins into small vesicles within multivesicular bodies (MVBs)64–66. When the MVBs fused with the cell surface, the small vesicles were released66. Interestingly, those EVs included Hsc70, the constitutively synthesized isoform of Hsp7067. That fact suggested to one of us (Tytell) that there might be some similarity between the microvesicle production in reticulocytes and the mechanism used by adaxonal glia in the squid axon to transfer proteins to the axon. It stimulated work reported a few years later that provided evidence that glial-derived vesicles were transferred into the axoplasm of the squid axon29.\n\nDuring the late 1980s and 1990s, work on membrane-bound vesicle release from cells increased greatly and the term exosome was applied more widely. However, released vesicles represent a very heterogeneous group, leading to attempts to classify them based on how they are produced, released, and isolated68. As reviewed in Smith et al.69, there are three groups of EVs in addition to the 30–100 nm vesicles commonly referred to as exosomes: (1) shedding vesicles that arise from blebbing from the cell surface as in apocrine secretion referred to earlier; (2) non-infectious retrovirus-like particles containing a subset of retroviral proteins; and (3) membrane-bound structures arising from apoptotic cells. Additional reviews by Théry70 and Harding et al.64 focusing on exosomes, showed that a huge variety of cytoplasmic and membrane components, including mRNAs and small, noncoding RNAs, could be released via exosomes from a variety of cells, and that the process of release is under the control of several of the Rab GTPases. This complexity has led to debates about the appropriate nomenclature for these structures, so we will use exclusively the acronym EVs to refer to them, as recommended by Gould and Raposo68. The diverse group of molecules found in EVs are generated by many different cell types that contain a common set of molecules plus unique proteins that are associated with cell type-related functions71. The common proteins include the tetraspanins, CD9 and CD63, that are involved in the biogenesis of exosomes and represent biomarkers for these organelles. Hence, they are often used for their selective isolation. Tetraspanins are a family of 30 proteins and specific members of this protein family can differ in exosomes that are derived from different tissues72. They consist of four transmembrane domains that form a tertiary structure believed to cluster proteins required for intraluminal vesicle (ILV) formation, an intermediary step in EV generation. Several other proteins typically found in EVs include Tsg101, Alix, Rab-GTPases, and annexins71–73. The formation of EVs is a complex process, beyond the scope of this review, and excellent, detailed discussions about this process can be found elsewhere70,71,74–77. In brief, EVs may be derived from the cell’s endosomal network that sorts ILVs into late endosomes, also referred to as MVBs. The sorting machinery involves an endosomal component known as the endosomal sorting complex (ESCRT), responsible for various aspects of the transport and sorting of the exosomal cargo76. The MVBs containing ILVs fuse with the plasma membrane and release the exosomes into the extracellular space where they can interact with various target tissues.\n\nAlthough the study of endosomal functions in the nervous system is still nascent, there is considerable evidence that EVs play significant roles as vehicles for communication in both directions between neurons and glia78. Both of these cells secrete exosomes and each cell type in the nervous system produces distinctive exosomes78. For example, astrocyte-derived exosomes contain Hsp/Hsc70, various growth factors such as fibroblast growth factor (FGF)-2 and vascular endothelial growth factor (VEGF), and angiogenic factors. Oligodendroglial exosomes contain various myelin proteins such as 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) and proteolipid protein (PLP), Hsp-70, -71, -90, factors that inhibit myelin formation, trophic factors for axonal integrity, various Rab proteins, and small RNAs. Microglial exosomes contain proinflammatory cytokines, interleukin-1beta, P2X7 receptors, major histocompatibility complex (MHC) class II, and various degradative enzymes75,78,79. In addition to the sorting and secretion of normal proteins to glial exosomes, they can accumulate and secrete abnormal molecules under pathological circumstances, serving as “double-edged swords” in being propagators of neurodegenerative disease and serving as biomarkers for medical diagnoses69,75,78,80,81. Recently, this pathological function has been a research focus in cancer82–86, as well as in the nervous system in glioblastoma69,87–89. Prion propagation has also been attributed, in part, to exosomes90,91.\n\nThus, exosome release represents a still-unfolding story of cell-to-cell communication with potentially widespread effects on target cells. Especially relevant to this review is the demonstration by Lancaster and Febbraio92,93 that it was via exosomes that Hsp70 was released from a variety of cultured cell types, including human peripheral blood monocytes. They showed that heat stress of 1 hour at 43°C increased significantly the Hsp70 content of those exosomes, although it did not alter the rate of exosomal release. That observation suggested that Hsp70 content of exosomes reflects the relative abundance of the protein in the cell cytoplasm. Whether Hsp70 may be specifically directed to exosomes in stressed cells remains unknown.\n\n\nNeuroprotective functions of exosomes and Hsps\n\nMany neurons, especially those with axons that are hundreds to thousands of times the diameter of the soma of origin (that is, centimeters or more in length), lack the typical stress response seen in most other cells. They do not increase their synthesis of Hsps in response to the level of metabolic stress typically encountered in an organism, such as fever-level hyperthermia (typically up to 45°C) or hypoxia94,95. This aberrant stress response has been considered paradoxical, as the vulnerability of neurons to such stresses is well known. Recently, differential regulation between glia and neurons of the heat shock factor, HSF1, binding to the gene promoter, was shown to underlie this distinction96. Glial and other non-neuronal cells like ependymal cells, however, have the typical stress response, increasing the synthesis of Hsp70 and some of the other Hsps markedly94,95,97–101. Nonetheless, neurons do become more stress resistant despite this deficient stress response if Hsp70 is present in extracellular fluid (reviewed in 102; see also 97,103,104). The fact that soluble Hsp70 has a domain that allows it to interact with and pass through the cell membrane may be another way that glia contribute to neuronal acquisition of stress tolerance105. However, because it seems likely that exosomes are the predominant means by which glia and other cells release their Hsp70, we suggest that the uptake of glial EVs containing Hsp70 is the likely way that neurons benefit from the robust glial stress response. In fact, several other reviews and reports78,99,106,107 strongly support this hypothesis and provide evidence for glial EVs supplying a variety of molecules promoting neuron function even under nonstressful conditions79,100,107,108.\n\n\nConclusions and future directions\n\nDespite the explosion of work on EVs in the last few years, a fundamental understanding of how these and other microvesicles interact with target cells such as neurons has only recently begun to be elucidated. There are a number of possible mechanisms by which neurons and other target cells could internalize these small EVs. Frühbeis et al.78,108 have reported that at least one process involves dynamin-dependent endocytosis. Although that process would place the exosome’s contents within a membrane-bound organelle inside the cell and thus would make them inaccessible to the cytoplasm, they showed that some of the EV components were functional within the neurons in a way that required access to the cytoplasm. One such example is the acquisition of glial Hsp70 by neurons leading to increased neuronal stress tolerance108. That suggests that the exosomal Hsp70 must gain access to the neuronal cytoplasmic compartment so that it can protect and refold cytoplasmic proteins in danger of denaturation and aggregation. Future research should focus on ways to follow exosomal contents so that we can understand how the exosomal cargo enters the target cell cytoplasm. As shown in Figure 1, we suggest several ways cytoplasmic localization of exosomal contents could happen: (1) by an intracellular membrane fusion event analogous to exocytosis of secretory vesicles; (2) by fusion of an exosome with the plasmalemma; or (3) by passage of exosomal molecules through membranes, either directly (as is possible for Hsp70105) or via carriers that are analogous to the way cytoplasmic proteins destined for the mitochondrial matrix are carried through the two mitochondrial membranes, a process that, coincidently, is dependent on Hsp70109.\n\nPanel A: An unknown mechanism allows Hsp70 (the stress-inducible form) or Hsc70 (the constitutively synthesized form), represented by asterisks, to become concentrated in secretory vesicles (hereafter, the proteins will be jointly designated as Hsp/c70). Arrow 1 indicates conventional exocytosis of Hsp/c70 into the extracellular space. Evidence for that process is limited110. Arrow 2 indicates interaction of the extracellular Hsp/c70 with the axonal membrane, reflected by a change in its shape, followed by diffusion into the axoplasm, where it is free to interact with other cytoplasmic components. This possibility must be considered because Hsp/c70 is known to interact with membrane phospholipids (reviewed in De Maio et al.111) and to include an amino acid sequence permitting passage through cell membranes105). Arrow 4 indicates uptake of Hsp/c70 by conventional endocytosis, after which it is present in endocytotic vesicles. From there it may diffuse through the endosomal membrane to enter the cytoplasm or it may remain inside as the endosome cycles through the endolysosomal pathway of the neuron. How that process might affect stress tolerance is unknown. Panel B: Depiction of two ways that exosomes containing glial cytoplasmic Hsp/c70 (4-pointed stars) could gain access to the cytoplasm of the axon. On the left side, arrow 1 indicates conventional apocrine secretion, in which small vesicles containing a mixture of cytoplasmic constituents bud off the cell surface. Alternatively, some of the cytoplasm of the donor may be enclosed within a vesicle by the process of autophagy (arrow 4). The multivesicular body resulting from autophagy may then be released by the glial cell via exocytosis (arrows 5 and 6). Thus, both apocrine secretion and exosome release yield the same result, a membrane-enclosed vesicle containing cytoplasmic constituents in the extracellular space. The released vesicle then may interact with the axon in either of two hypothetical ways. It may fuse with the plasmalemma of the recipient cell, releasing its contents, including Hsp/c70, into the neuron’s cytoplasm (indicated by arrows 2 and 3). Alternatively, the released vesicle may be phagocytosed by the axon, forming another multivesicular body. Then, the inner vesicle membrane may fuse with the outer vesicle membrane in a process analogous to exocytosis, releasing the Hsp/c70 and other cytoplasmic constituents from the glial cell-derived vesicle into the cytoplasm of the axon (indicated by arrows 8–10).\n\nAnother intriguing area just beginning to be explored is to determine how to engineer exosomes and other EVs as therapeutic vehicles for targeted drug, gene, protein, or lipid delivery71,73,75,81. Recently, Yuyama et al.112 have shown that neuronal exosomes that contained abundant glycosphingolipids could sequester intracerebral amyloid-β peptide in the brains of amyloid precursor protein transgenic mice and decrease amyloid-β and amyloid depositions in the brain. Thus, in addition to furthering our understanding of the long-standing puzzle of how glia support neurons and their axons, a better understanding of the function of exosomes and other EVs may provide new tools for combatting neurodegenerative diseases.", "appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nHarold Gainer’s contribution to this research was supported by the Intramural Research Program of the NINDS, NIH.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThis review was initiated while Michael Tytell and Harold Gainer were library researchers at the Marine Biological Laboratory, Woods Hole, MA 02543, July to September, 2015. The authors thank the reviewers, especially Eva-Maria Kramer-Albers and Scott T. Brady, for their careful reading of the manuscript and thoughtful comments and the editorial staff of F1000 for their help in preparing it for publication.\n\n\nReferences\n\nBullock T, Horridge G: Structure and Function in the Nervous Systems of Invertebrates. San Francisco, London. 1965; 2. . Reference Source\n\nKandel E, Schwartz J, Jessell T, et al.: Principles of Neural Science. 5 ed. New York: W H Freeman; 2012. Reference Source\n\nDroz B, Leblond CP: Axonal Migration of Proteins in the Central Nervous System and Peripheral Nerves as Shown by Radioautography. J Comp Neurol. 1963; 121(3): 325–346. PubMed Abstract \| Publisher Full Text\n\nGrafstein B, Forman DS: Intracellular transport in neurons. Physiol Rev. 1980; 60(4): 1167–1283. PubMed Abstract\n\nLasek R: Axoplasmic transport in cat dorsal root ganglion cells: as studied with [3H]-L-leucine. Brain Res. 1968; 7(3): 360–377. 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Philos Trans R Soc Lond B Biol Sci. 2014; 369(1652): pii: 20130516. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nSimons M, Raposo G: Exosomes--vesicular carriers for intercellular communication. Curr Opin Cell Biol. 2009; 21(4): 575–581. PubMed Abstract \| Publisher Full Text\n\nThéry C, Zitvogel L, Amigorena S: Exosomes: composition, biogenesis and function. Nat Rev Immunol. 2002; 2(8): 569–579. PubMed Abstract \| Publisher Full Text\n\nFrühbeis C, Fröhlich D, Kuo WP, et al.: Extracellular vesicles as mediators of neuron-glia communication. Front Cell Neurosci. 2013; 7: 182. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBakhti M, Winter C, Simons M: Inhibition of myelin membrane sheath formation by oligodendrocyte-derived exosome-like vesicles. J Biol Chem. 2011; 286(1): 787–796. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nColeman BM, Hill AF: Extracellular vesicles--Their role in the packaging and spread of misfolded proteins associated with neurodegenerative diseases. Semin Cell Dev Biol. 2015; 40: 89–96. PubMed Abstract \| Publisher Full Text\n\nTsunemi T, Hamada K, Krainc D: ATP13A2/PARK9 regulates secretion of exosomes and α-synuclein. J Neurosci. 2014; 34(46): 15281–15287. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAnastasiadou E, Slack FJ: Cancer. Malicious exosomes. Science. 2014; 346(6216): 1459–1460. PubMed Abstract \| Publisher Full Text\n\nGreening DW, Gopal SK, Mathias RA, et al.: Emerging roles of exosomes during epithelial-mesenchymal transition and cancer progression. Semin Cell Dev Biol. 2015; 40: 60–71. PubMed Abstract \| Publisher Full Text\n\nMinciacchi VR, Freeman MR, Di Vizio D: Extracellular vesicles in cancer: exosomes, microvesicles and the emerging role of large oncosomes. 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PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation" }	[ { "id": "12597", "date": "22 Feb 2016", "name": "Peter W Baas", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12598", "date": "22 Feb 2016", "name": "Eva-Maria Krämer-Albers", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12599", "date": "22 Feb 2016", "name": "Scott Brady", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-205
https://f1000research.com/articles/5-204/v1	22 Feb 16	{ "type": "Review", "title": "Recent Advances in Urinary Tract Reconstruction for Neuropathic Bladder in Children", "authors": [ "Roberto I. Lopes", "Armando J. Lorenzo", "Roberto I. Lopes" ], "abstract": "Neuropathic bladder usually causes several limitations to patients’ quality of life, including urinary incontinence, recurrent urinary tract infections, and upper urinary tract damage. Its management has significantly changed over the last few years. The aim of our paper is to address some salient features of recent literature dealing with reconstructive procedures in pediatric and adolescent patients with lower urinary tract dysfunction.", "keywords": [ "urinary reservoirs", "continent", "urinary incontinence", "urinary bladder", "neuropathic", "fecal incontinence" ], "content": "Introduction\n\nSpinal dysraphism, including myelomeningocele, represents one of the most common permanently disabling birth defects in the United States, with an incidence of 30 cases in 100,000 live births1. “More than 90% of patients with spina bifida have resultant neuropathic bladder dysfunction, which can manifest as urinary incontinence, recurrent urinary tract infections and—in the most severe cases—upper urinary tract damage”2. Unfortunately, some degree of renal impairment is common, affecting up to 30% of adolescents with the condition3. Although most patients can be managed with medication (e.g. anticholinergics) and clean intermittent catheterization, lower urinary tract reconstructive surgery has been introduced and modified over the last few decades to address incontinence and prevent upper tract decompensation. Despite perceived benefits and after a fairly rapid uptake, the estimated number of augmentation cystoplasties performed in children in the United States has now decreased by 25% in the 2000s. The cause for this change is likely multifactorial, including better or earlier introduction of optimal medical management, but ultimately reflects an important change in practice patterns in the United States and may mirror trends in other parts of the world. It is tempting to consider the surgeries’ risk profile—with up to 30% of patients having a potential complication during hospitalization after augmentation cystoplasty—and the known long-term consequences of this procedure as the driving force behind this trend4.\n\nWhen considering any surgical intervention in patients with neuropathic bladder, evaluation of the patient’s clinical status according to a risk-stratified inventory is advised: one must a) confirm that the upper tracts are stable without new dilation, increasing renal echogenicity, or deteriorating corticomedullary differentiation; b) assess whether the child has been experiencing urinary tract infections (UTIs); and c) determine if urinary incontinence is becoming a concern that the child wishes to have addressed.\n\nUrodynamic or videourodynamic evaluations have proven to be of great value in quantifying bladder dysfunction, helping guide therapy for socially unacceptable incontinence and/or potential renal insults, as well as evaluating the outcome of resulting procedures and interventions. Importantly, a significant proportion of patients with spina bifida have reduced bladder capacity with different degrees of impaired compliance. The most worrisome situation, a “high-pressure” bladder, is characterized by increased leak point pressure, reduced bladder compliance, and detrusor overactivity, a situation that—if untreated—often leads to complications down the road. A detrusor leak-point pressure (DLPP) >40 cm H2O, a bladder compliance of <9 mL/cmH2O, and evidence of hypercontractile detrusor are all factors that carry some value in predicting the risk of upper urinary tract dysfunction in children with neuropathic bladder5.\n\nFirst-line therapy for reduced bladder capacity and/or high-pressure bladder is anticholinergic medication, usually coupled with clean intermittent catheterization. If this approach fails or is not tolerated by the patient, second-line options include a variety of procedures such as botulinum toxin injection, electrical stimulation therapy, and bladder autoaugmentation6. One exciting option is the direct injection of botulinum toxin in the detrusor muscle as a means to quench detrusor overactivity and improve compliance. Over the past few years, this option has gained popularity7,8. Intra-detrusor OnabotulinumtoxinA (OnabotA) injections have been selectively offered at our institution for cases in which maximal anticholinergic therapy failed or was not tolerated. Thus far, it has shown significant improvement in symptoms, bladder capacity, and compliance, effectively avoiding or delaying the need for augmentation at the expense of regular procedures to deliver the medication9.\n\nIn refractory cases, bladder neck reconstruction (BNR), bladder augmentation, continent diversion (CD), and bladder neck closure (BNC) are offered, with the goal of creating a large capacity and highly compliant reservoir that can be easily accessed (for catheterization) without leakage at expected volumes for age. These surgical approaches may be necessary in 5% to 20% of patients10,11. Additional procedures are often required. For example, creation of a catheterizable channel (with appendix or reconstructed, tubularized bowel) is offered as a means for providing more convenient access and/or a reliable entry point when catheterization per urethra is difficult or impossible. Similarly, bilateral high-grade vesicoureteral reflux in patients with neuropathic bladder can persist after bladder augmentation in up to half of patients. Many of these develop pyelonephritis during follow-up, even while taking antibiotic prophylaxis. Therefore, at the time of bladder augmentation for noncompliant neuropathic bladder, concomitant anti-reflux surgery should be considered for children with high-grade reflux12. In severe cases of high-pressure non-compliant bladders that are causing renal decompensation, especially in the setting of kids with severe disabilities who would require constant care and assistance, and for whom an easy, failsafe method of bladder management is preferred, a vesicostomy can be used as a temporizing factor for some and a permanent solution for others.\n\nWithin this framework, herein we will discuss some salient features of recent literature dealing with reconstructive procedures in pediatric and adolescent patients with lower urinary tract dysfunction.\n\n\nAugmentation cystoplasty\n\nBladder augmentation and related urinary diversion techniques aim to dramatically reduce pressure to the upper tracts, prevent further renal damage, and aid in continence. However, these procedures have several drawbacks. For patients who undergo ileal loop urinary diversion, limitations include altered body image, management of an external appliance, and the potential for recurrent pyelonephritis, nephrolithiasis, and delayed anastomotic stricture. Most patients who undergo bladder augmentation require intermittent catheterization, with its own set of challenges. More importantly, long-term complications of bladder augmentation include metabolic derangement, bladder stones, recurrent UTI, bladder perforation, and an ill-defined increased risk of malignancy. The choice between urinary diversion and bladder augmentation is complex for surgeons and patients. Surgeon factors include comfort with the surgical technique and resources for subsequent management. For patients and their families, important considerations include body image, social and cultural issues, ability to perform intermittent catheterization, anticipated compliance with long-term follow-up, need for indefinite monitoring, and unknown problems as decades go by exposing intestinal epithelium to urine.\n\nIn one of the largest studies comparing these two techniques, bladder augmentation was performed in an estimated 3403 patients and ileal loop diversion in 772 patients with spina bifida between 1998 and 2005. Patients undergoing bladder augmentation were younger (mean age 16 vs. 36 years, p <0.001), more often male (52% of bladder augmentations vs. 43% of urinary diversions, p = 0.02), and privately insured (46% vs. 29%, p <0.001) compared to those undergoing urinary diversion. “Furthermore, patients undergoing urinary diversion required more health care resources, with significantly longer hospital stays, higher total charges and more use of home health care after discharge home”13. This highlights important differences in demographics and health care resource utilization between populations exposed to different surgical strategies and might indicate that children getting urinary diversions are generally sicker at baseline evaluation.\n\nChildren who underwent augmentation cystoplasty identified in the Pediatric Health Information System over a decade were assessed to evaluate their surgical outcomes. A total of 2831 augmented patients were assessed and 10-year cumulative incidence ranges for the following outcomes and procedures were achieved: bladder rupture (2.9–6.4%), small bowel obstruction (5.2–10.3%), bladder stones (13.3–36.0%), pyelonephritis (16.1–37.1%), and need for cystolithopaxy (13.3–35.1%) and re-augmentation (5.2–13.4%). “The development of chronic kidney disease was strongly associated with a diagnosis of lower urinary tract obstruction (HR 13.7; 95% CI 9.4–19.9). Bladder neck surgery and stoma creation at time of augment were associated with an increased hazard of bladder rupture (HR 1.9; 95% CI 1.1–3.3) and bladder stones (HR 1.4; 95% CI 1.1–1.8) respectively”. Results from this large cohort can be used to counsel patients and families about expectations for surgical intervention, including those that carry important morbidity, such as bladder perforation14. Finally, the risk of malignancy development has been of concern to many but appears to be <5%15,16. Obviously, this may dramatically change as the patient population ages and is exposed to carcinogenic stimuli (such as smoking and chronic irritation). The issue of malignancy should be further considered in all patients with neuropathic bladder, with or without augmentation, and can be the end-result of catheterization, infections, and/or colonization. As recently reported by Husmann et al., other factors (such as immunosuppression) can drive the risk of carcinogenesis in a population already at risk due to their lower urinary tract dysfunction15.\n\nWith the widespread interest in minimally invasive surgery, robotic augmentation and appendicovesicostomy has been recently described, isolated to centers with extensive expertise. Although feasible, the surgery does appear somewhat cumbersome and time-consuming. The intervention clearly tried to mimic the surgical steps of its open counterpart. For augmentation cystoplasty, the patient is placed in the Trendelenburg position. An umbilical 12 mm trocar is placed, followed by two 8 mm robotic ports and two assistant ports. After pneumoperitoneum creation, the bladder, small bowel, and appendix are assessed. Stay sutures are placed 20 cm apart on the portion of the ileum that will be used for augmentation, 20 cm from the ileocecal junction. Entero-entero anastomosis is performed in an end-to-end fashion followed by mesenteric window closure. For appendiceal dissection, a stay suture in the tip is used and a window is created in the mesentery of the appendix, making sure that its blood supply is preserved. The appendix is detached from the cecum, with the latter closed in two layers. Detrusorotomy is performed after distension of the bladder with saline, and the tip of the appendix is spatulated and anastomosed to the bladder mucosa after a 1 cm opening of its more distal aspect. Appendicovesical anastomosis is performed in a continuous fashion over a 8 Fr feeding tube placed through the appendix, and after that a tunnel to avoid reflux is created by the imbrication of the detrusor muscle over the appendix. The bladder is bivalved to receive the detubularized ileal segment, which is anastomosed as in open augmentation. An 18 Fr Foley catheter is introduced into the bladder before completing the ileovesical anastomosis. The appendix can be exteriorized through the umbilicus or the lower quadrant of the abdomen. The augmented bladder is drained with suprapubic and urethral catheters until ready to start clean intermittent catheterization through the appendicovesicostomy 7–10 days post op17–19. The reported average operative time was 8.4 hours (range 6–11 hours) and no major intraoperative complications were encountered. Perioperatively, patients required oral analgesia for 24–36 hours, started on a liquid diet after 7.5 hours (range 6–10 hours), went on a regular diet after 24 hours (range 12–36 hours), and were discharged home within 7 days. All patients now have day and night time continence without UTIs, and bladder capacity between 250 and 450 mL. While longer follow-up will be necessary to see if these results are durable, this series demonstrates that robotic alternatives are safe and feasible in the short term, with the possible added benefits of reduced analgesia and recovery time, along with aesthetic benefits. Rightfully, it is important to question all these outcomes against open surgical procedures with less expensive resource utilization and acceptable, well-described, longer-term outcomes.\n\n\nGastric augmentation\n\nGastric augmentation seemed like a great idea when originally introduced, particularly for patients with chronic renal insufficiency, in which the acid-base balance changes expected from recurrent drainage of acid gastric mucosa outputs would benefit the patient. Unfortunately, after long-term follow-up, complications can be expected in over half of patients. Malignancies have developed in the reservoir in some patients in as little as a decade after gastrocystoplasty20. We currently do not recommend the use of gastric segments for reconstruction of the lower urinary tract due to the high incidence of reoperations and complications. In patients in whom gastric segments were used in the past for lower urinary tract reconstruction, regular surveillance and close follow-up are strongly advocated, even though it is unknown if any preventive or screening strategies are of value.\n\nMetabolic complications with the use of stomach for urinary reconstruction have been previously described. Abnormalities directly related to the secretion of hydrochloric acid by the gastric patch incorporated in the urinary tract include the hematuria-dysuria syndrome, hypochloremic metabolic alkalosis, and hypergastrinemia. The hematuria-dysuria syndrome is a unique complication of the use of gastric tissue for lower urinary tract reconstruction. The secretion of acid can irritate the bladder and urethra, and the reported incidence can be as high as 36%. Severity of symptoms varies, and many cases are relatively minor and can be controlled with H2 histamine blockers, proton pump inhibitors, and increased hydration and frequency of catheterization. In unresponsive cases, removal of gastric tissue is necessary20.\n\n\nBiomaterials\n\nThe idea of using a readily available “off-the-shelf” material has attracted investigators for decades. Only a few clinical studies have been reported, showing mostly disappointing results, employing biological materials such as dura mater or small intestinal mucosa21. When contrasted with the use of autologous intestinal tissue, these alternatives have not shown significant benefit and can lead to failure or complications.\n\n\nBioengineered bladder\n\nExperimental efforts to construct a tissue-engineered bladder with a scaffold seeded with cultured cells previously obtained from the patient’s bladder have been reported, culminating in a somewhat controversial report of a few patients with myelomeningocele, showing limited application and mostly disappointing results22. The current techniques for autologous cell-seeded biodegradable scaffolds do not appear to improve bladder compliance or capacity in a clinically superior way, and serious adverse events appear to surpass acceptable safety standards23.\n\nAn important problem with contemporary approaches to tissue engineering and cell therapy for urinary tract reconstruction is the requirement for invasive tissue biopsies to obtain autologous cells. Aside from the need to accept the morbidity and exposure to anesthesia for such procedure, there is a theoretical problem with employing cells whose genetic and epigenetic footprint may have been altered from development in a diseased organ. As an alternative, a urine-isolated subpopulation of cells with progenitor cell features and the potential to differentiate into several bladder cell lineages can be employed. These urine-derived cells could serve as an alternative source for urinary tract tissue engineering and reconstruction24. The ultimate value is speculative, and animal and human studies are clearly needed. This exciting field is growing at an exponential pace. Even though in clear need of a breakthrough, the promise fuels hope for patients and families.\n\n\nButtons\n\nButtons might be an alternative to patients who are not amenable to augment/diversion and/or clean intermittent catheterization. Two different types of button can be used: the Mic-Key button (Ballard Medical Products) and the Mini balloon button (Applied Medical Technology). Both are silicone devices. They consist of an internal portion, resembling the tip of a Foley catheter, with an inflatable balloon to create a self-retaining mechanism against the abdominal wall. The external portion comprises a flat button that sits on the skin surface. It contains a valve to prevent leakage unless the drainage adapter is in place. The devices are available in a range of sizes (12–24 Fr) and lengths (0.8–5.0 cm), allowing the catheter to be individualized based on the patient’s characteristics and growth over time. Although an interesting option, there is a paucity of data suggesting superiority over a suprapubic catheter or other forms of diversion. Furthermore, its use does not address problems with capacity or compliance, only access for bladder drainage25.\n\n\nCatheterizable channels\n\nThe Mitrofanoff principle for creating a continent, catheterizable stoma using the appendix has been a mainstay in the armamentarium of the pediatric urologist to provide access to the bladder either with or without augmentation since its description and popularization in the 1980s26. The use of a transverse tubularized bowel segment, as described by Yang and Monti27, and further modified by Casale28, has now become the preferred option when the appendix is unavailable. Irrespective of how the channel is fashioned, patients and families should be aware about the potential need for reinterventions, which persists even during long-term monitoring29.\n\nSimilar to robotic augmentation cystoplasty, robotic appendicovesicostomy is gaining popularity. The patient is placed in the Trendelenburg supine position, and an 8 or 12 mm camera port is placed at the umbilicus with two 8 mm working ports and one 5 or 12 mm assistant port. The appendix is identified and separated from the cecum with an articulating 55 GIA™ vascular stapler and carefully mobilized on its blood supply. An approximately 4 cm posterior detrusorrhaphy is created, the appendix is laid in the channel, and the bladder is closed over it in an interrupted or running fashion with 3-0 absorbable suture. The bladder is hitched to the abdominal wall and the appendix brought to the umbilical port site, where the stoma is matured. A catheter is left in the appendicovesicostomy channel for approximately 4 weeks30. Comparison of robotic and open appendicovesicostomy revealed no significant difference in the number of acute complications or reoperations between groups. However, the nature and timing of complications differed between groups, being earlier in the robotic approach and later in open surgery31–33. Statements regarding equivalency or slight (statistically significant) benefit between open and robotic procedures call into question the presence of any clinical benefit when considering resource utilization and expenses. As a bridge, laparoscopic Mitrofanoff has also been reported to have good results, being a feasible, safe, and effective technique associated with low morbidity34. In some centers, including ours, a mixed approach has also been used, employing laparoscopy (with ports placed in the umbilicus and along the planned Pfannenstiel-type incision) to identify and mobilize the appendix towards the pelvis, followed by open detachment from the cecum and anastomosis to the bladder (Figure 1A and B).\n\nLaparoscopic-assisted Mirofanoff channel: A. Laparoscopy (with ports placed in the umbilicus and along the planned Pfannenstiel-type incision) to identify and mobilize the appendix towards the pelvis; B. Detrusorotomy and confection of Mitrofanoff channel.\n\n\nSlings\n\nPatients presenting urethral intrinsic sphincteric insufficiency (low DLPP) can benefit from surgically increasing resistance as part of urinary tract reconstruction. On comparison, patients undergoing slings with and without augmentation appear similarly successful in achieving improved continence, with patients undergoing augmentation having a longer interval between catheterization and requiring fewer anticholinergics. However, this has to be interpreted with caution, as the patient characteristics in these groups are bound to be different. Health-related quality of life responses revealed that both cohorts were similarly satisfied with the outcomes35. There are some potential differences in outcomes related to the type of procedure performed. For example, patients undergoing Leadbetter-Mitchell procedure plus fascial sling may be less likely to require pads postoperatively than those having a sling alone. Other BNR strategies may be associated with difficulty catheterizing per urethra, thus are often performed in conjunction with a catheterizable channel. Ultimately, this highlights the possibility that some of these procedures are in effect acting as a BNC. In addition, the long-term implications of these procedures are not well defined, and there are evolving challenges that present over time. The best example of such a statement is the disappointing development of problems after BNR without augmentation in a cohort of patients initially reported to enjoy great outcomes. After a mean follow-up of almost 5 years, the updated series described over 50% need for additional continence surgery augmentation cystoplasty, along with a high proportion of vesicoureteral reflux, hydronephrosis, and newly diagnosed or worsening renal scarring36. These findings begin to resemble other cautionary reports addressing issues over prolonged monitoring37, highlighting the need for close monitoring and critical assessment of successful interventions reported after relatively short follow-up.\n\nOf all the described procedures and their variations, bladder neck sling cystourethropexy is a commonly used one to correct relative sphincter deficiency in children with spinal dysraphism. Various modifications of the procedure have been made, but as a common theme they involve circumferential dissection of the bladder neck and proximal urethra. Robotic-assisted laparoscopic placement of a bladder neck sling has been recently performed for two female patients with intrinsic sphincter deficiency but adequate bladder compliance. Both procedures were completed intracorporeally. The mean blood loss was 20 mL. The mean operative time was 189 minutes. No intraoperative or postoperative complications occurred. The mean hospital stay was 3 days (range 2–4). The follow-up ranged from 13 to 22 months. Postoperative studies revealed continued low-pressure, compliant bladders and stable upper tracts. At last follow-up, the two patients were using catheterization without difficulty and were continent38. A step-by-step description of the technique and recommendations on robotic instrumentation have been recently published39.\n\n\nBladder neck injections and reconstruction\n\nDeflux® (Dx/HA) bladder neck injection after slings results in modest and often disappointing results. For example, in one series, dryness was achieved in only 25% of patients after failed sling. Second injections after either failure rarely achieve dryness and are hard to justify40. Other groups have reported that endoscopic treatment of neurogenic urinary sphincter insufficiency with Dx/HA is effective in about half of patients41. In general, failure should call for attention to bladder dynamics, a potential culprit for persistent incontinence, and avoid restricting future interventions solely to the bladder neck.\n\nAs previously mentioned for slings, isolated bladder outlet procedures for neurogenic incontinence (including the use of artificial sphincters) portend a poor long-term outcome. During long-term follow-up, these patients often require additional interventions (such as augmentation cystoplasty) despite diligent use of anticholinergic medications and strict catheterization. Unfortunately, preoperative urodynamic evaluation does not appear to predict the need or timing for initial bladder outlet procedure or future augmentation cystoplasty, supporting the dynamic picture presented by neuropathic dysfunction, which can change over time and in response to surgical interventions42.\n\nRecently, robotic Leadbetter bladder neck surgical repair has been reported. The patient is placed supine on a surgical beanbag positioner. A 14 F urethral catheter is placed. Pneumoperitoneum is created. An umbilical 12 mm port for the robotic camera is employed, with two 8 mm and one 12 mm additional working ports. A crescent-shaped incision is made posterior to the bladder to drop the rectum in males or vagina in females. The peritoneum is incised and space of Retzius developed. On the posterior aspect, the rectovesical space is developed, from which point the aforementioned tunnelers are advanced ventrally into the developed space of Retzius. The bladder is dropped anteriorly, and the suspensory puboprostatic ligaments are divided. A dorsal vein stitch is placed before cutting down to the catheter and unroofing the proximal urethra and bladder neck to the level of the interureteric ridge. The ureteral orifices are identified, aided by the intravenous injection of indigo carmine. After exchanging the Foley catheter for a 5 Fr feeding tube, the bladder neck/urethra is retubularized in two layers. Afterwards, the tunneling devices are identified and employed to tightly wrap a 360 degree sling that is subsequently attached to the pubic bone using screws from a hernia tacker43.\n\nPostoperative bladder capacity was reported as adequate and augmentation was not deemed necessary. Mean DLPP was 29 cm H2O. Mean operative time was close to 8 hours (range 5:56–12:18), including time for appendicovesicostomies that were also performed for clean intermittent catheterization. Mean length of stay and blood loss were 85.7 hours and 117.8 mL, respectively. Postoperatively, all patients were completely dry on clean intermittent catheterization and anticholinergics. This initial series of robot-assisted appendicovesicostomy with BNR and sling placement demonstrates the procedure to be feasible and safe. Needless to say, there is little theoretical basis to suggest that robotic procedures would be less likely to trigger deterioration during follow-up, thus long-term monitoring is required irrespective of how the bladder neck intervention is carried out.\n\n\nBladder Neck Closure\n\nBNC is often seen as a last resort. It is sometimes necessary to improve quality of life in severe refractory cases. In boys, the bladder is transected just cranial to the prostate, after individualizing the neurovascular bundles. In girls, transection is done between the bladder neck and urethra. The bladder is then mobilized on the dorsal side to the level of the ureteric orifices. The bladder neck and urethral stump are then closed. In order to avoid contact, the bladder stump is fixed ventrally to the pubic bone and urethral stump dorsally. If possible, an omental flap might be brought in between the two stumps44. BNC in conjunction with enterocystoplasty and Mitrofanoff diversion is an effective means of achieving continence in complex cases as a primary or secondary therapy45.\n\n\nArtificial urinary sphincter\n\nAnother option to address urinary intrinsic insufficiency is the artificial urinary sphincter. Although it provides a good rate of continence, complications are frequent, leading to removal in 20% of cases. Critics highlight the added issue of mechanical dysfunction. Over time, only a limited number of patients can empty the bladder without clean intermittent catheterization, which is touted as one of the great benefits of the intervention46.\n\n\nFecal incontinence\n\nFecal incontinence has a significant impact on quality of life, leading to loss of self-esteem, social isolation, and depression. Initial management of neurogenic bowel includes stool softeners, bulking agents, and − if these approaches fail or prove insufficient − timed evacuation with suppositories and retrograde enemas. However, self-administering enemas can be challenging for patients with limited dexterity and challenge independence. Surgical options to address this issue include the Malone antegrade continence enema (MACE) and cecostomy button; these allow for direct administration of fluid into the bowel, which is accessed through the abdominal wall.\n\nThe “MACE principle” was first introduced in the 1990s47. Significant improvements in quality of life following this procedure have been observed. The intervention follows the Mitrofanoff principle and involves creating an appendicostomy as a conduit for antegrade enema administration. A percutaneous technique, with image-guided insertion of a cecostomy tube for similar antegrade enema administration, was established in 1996.\n\nLaparoscopically assisted MACE is now performed in most pediatric referral centers. A 5 mm scope is inserted through the umbilicus and two ports are placed in the left lower quadrant. The appendix is mobilized, brought to the umbilicus, and fixed to the abdominal wall or right lower quadrant.\n\nFor cecostomy button placement, a 20 Fr Foley catheter is placed per rectum and the colon is insufflated with air. A suitable access site overlying the cecum is selected and ultrasound is used to confirm the safety of the access. Two suture anchor needles are used to secure the cecum. The initial tube is placed in one of the two access sites. The tract is dilated to approximately 8 Fr and an 8 Fr Dawson-Mueller Mac-Loc® catheter is placed. Six weeks later, the second portion of the procedure is completed, when the catheter is changed to a Chait Trapdoor™ cecostomy catheter, under fluoroscopic guidance over a wire. General anesthesia is usually required. Yearly exchanges of the cecostomy catheters are recommended. Young children require general anesthesia for the initial tube changes, while older patients tolerate it without any sedation.\n\nFecal continence rates of up to 85% for MACE and 90% for cecostomy tubes have been reported. Mean length of hospital stay for patients undergoing cecostomy vs. laparoscopically assisted MACE was similar. Complications included stomal pain (23% of patients) and difficulty with catheterizing (19%) following MACE, and difficulty flushing (26%) following cecostomy. There were no significant differences between MACE and cecostomy button with respect to fecal continence or complication rates. Each approach poses unique challenges, suggesting that physicians, patients, and families need to understand the differences to make an individualized choice48.", "appendix": "Competing interests\n\n\n\nThe authors declare that they 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\nLloyd JC, Wiener JS, Gargollo PC, et al.: Contemporary epidemiological trends in complex congenital genitourinary anomalies. J Urol. 2013; 190(4 Suppl): 1590–1595. PubMed Abstract \| Publisher Full Text\n\nSnow-Lisy DC, Yerkes EB, Cheng EY: Update on Urological Management of Spina Bifida from Prenatal Diagnosis to Adulthood. J Urol. 2015; 194(2): 288–296. 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J Pediatr Urol. 2013; 9(3): 278–282. PubMed Abstract \| Publisher Full Text\n\nAlova I, Margaryan M, Bernuy M, et al.: Long-term effects of endoscopic injection of dextranomer/hyaluronic acid based implants for treatment of urinary incontinence in children with neurogenic bladder. J Urol. 2012; 188(5): 1905–1909. PubMed Abstract \| Publisher Full Text\n\nDave S, Salle JL: Current status of bladder neck reconstruction. Curr Opin Urol. 2008; 18(4): 419–424. PubMed Abstract \| Publisher Full Text\n\nBagrodia A, Gargollo P: Robot-assisted bladder neck reconstruction, bladder neck sling, and appendicovesicostomy in children: description of technique and initial results. J Endourol. 2011; 25(8): 1299–1305. PubMed Abstract \| Publisher Full Text\n\nDe Troyer B, Van Laecke E, Groen LA, et al.: A comparative study between continent diversion and bladder neck closure versus continent diversion and bladder neck reconstruction in children. J Pediatr Urol. 2011; 7(2): 209–212. PubMed Abstract \| Publisher Full Text\n\nKavanagh A, Afshar K, Scott H, et al.: Bladder neck closure in conjunction with enterocystoplasty and Mitrofanoff diversion for complex incontinence: closing the door for good. J Urol. 2012; 188(4 Suppl): 1561–1565. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nCatti M, Lortat-Jacob S, Morineau M, et al.: Artificial urinary sphincter in children--voiding or emptying? An evaluation of functional results in 44 patients. J Urol. 2008; 180(2): 690–3; discussion 693. PubMed Abstract \| Publisher Full Text\n\nMalone PS, Ransley PG, Kiely EM: Preliminary report: the antegrade continence enema. Lancet. 1990; 336(8725): 1217–1218. PubMed Abstract \| Publisher Full Text\n\nHoy NY, Metcalfe P, Kiddoo DA: Outcomes following fecal continence procedures in patients with neurogenic bowel dysfunction. J Urol. 2013; 189(6): 2293–2297. PubMed Abstract \| Publisher Full Text" }	[ { "id": "12592", "date": "22 Feb 2016", "name": "Aseem R. Shukla", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12593", "date": "22 Feb 2016", "name": "Dana Weiss", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-204
https://f1000research.com/articles/5-203/v1	22 Feb 16	{ "type": "Review", "title": "Developmental gene regulatory networks in sea urchins and what we can learn from them", "authors": [ "Megan L. Martik", "Deirdre C. Lyons", "David R. McClay", "Megan L. Martik", "Deirdre C. Lyons" ], "abstract": "Sea urchin embryos begin zygotic transcription shortly after the egg is fertilized. Throughout the cleavage stages a series of transcription factors are activated and, along with signaling through a number of pathways, at least 15 different cell types are specified by the beginning of gastrulation. Experimentally, perturbation of contributing transcription factors, signals and receptors and their molecular consequences enabled the assembly of an extensive gene regulatory network model. That effort, pioneered and led by Eric Davidson and his laboratory, with many additional insights provided by other laboratories, provided the sea urchin community with a valuable resource. Here we describe the approaches used to enable the assembly of an advanced gene regulatory network model describing molecular diversification during early development. We then provide examples to show how a relatively advanced authenticated network can be used as a tool for discovery of how diverse developmental mechanisms are controlled and work.", "keywords": [ "developmental gene regulatory networks", "sea urchin", "dgrn", "patterning", "morphogenetic cassetes" ], "content": "The sea urchin developmental gene regulatory networks\n\nDevelopmental gene regulatory networks (dGRNs) describe the sequential regulatory changes that specify and diversify the cells of an embryo. The genes included in dGRNs encode transcription factors, components of signal transduction pathways, and often effector genes as markers of differentiated cell states. Models of dGRNs are assembled on the basis of experimental perturbations of an embryo’s developmental program and are valuable for explaining how spatial and temporal information is encoded in a multicellular organism’s genome. dGRNs have the potential of providing a causal understanding of how upstream specification controls downstream events (i.e. differentiation or cell biological functions). As such, a dGRN can serve as a tool for developmental and cell biologists alike. This article describes how dGRNs are assembled using the sea urchin embryo as a model and includes recent insights into sea urchin development that have benefitted from the assembly exercise.\n\nThe early specification events of the sea urchin embryo have been extensively documented, resulting in increasingly well-understood dGRNs for each cell type. Sea urchin development is relatively simple, easy to observe, and experimentally tractable, and experimental outcomes are rapidly obtained. Prior to genome sequencing, a provisional sea urchin endomesodermal dGRN was assembled starting with a small number of transcription factors and signaling inputs1. In 2006, the sea urchin genome was sequenced2, and since that time, labs across the globe have added to and reinforced an understanding of the regulatory linkages in the dGRNs through gastrulation3–9. In contrast to typical “hairball” or “interactome” networks, where hypothetical interactions are based on statistical inference, each linkage of the sea urchin dGRN is based on several experimental approaches, spatial and temporal validation, and in many cases cis-regulatory analyses to confirm a direct regulatory connection. This effort has provided many valuable insights into network function and has led to novel explorations into mechanisms of sea urchin development. We begin by summarizing experiments that demonstrated how the sea urchin dGRN was, and continues to be, assembled. Then we briefly review how dGRNs have been used as tools to understand how embryonic patterning works, how morphogenesis is controlled, and how evolutionary processes have modified dGRNs.\n\n\nDevelopmental gene regulatory network assembly process\n\nGRNs reflect the relationship between genes in a system. In a graphic depiction of GRNs, the expression of a transcription factor or a component of a signal transduction pathway is represented as a node. The edges or connections between nodes reflect the regulatory relationship between nodes over time. In GRN models, an upstream regulator is drawn with an output that either activates or represses expression of a downstream gene. At any given time after development begins, many transcription factors are expressed and are under regulatory control by transcription factors upstream of them. Over time, this produces a regulatory network with many properties. At any given time, a cell in an embryo is controlled by the unique regulatory “state” of its GRN. All identical cells will tend to be regulated by the same GRN state, and as cells diverge from one another, their regulatory states change. Signaling molecules produced by one cell pass to adjacent or nearby cells where their inputs alter GRN states in recipient cells. Within a GRN state, it is possible to tease apart relationships of transcription factors and identify sub-circuits designed to accomplish the tasks of development. Among the tasks uncovered in the sea urchin dGRN are a number of properties that are commonly seen when network systems are studied in detail10–12. For example, a “double-negative gate” (repression of a repressor) was identified to initiate specification of a specific cell type4,13. “Spatial exclusion” sub-circuits were found in cells at the time these cells diverged from one another toward differentiation into distinct cell types; in each of the two nascent cell types, there was a sub-circuit to exclude the other14. “Community effect” signaling was identified such that Nodal reinforced Nodal signaling in neighboring ectodermal cells to maintain Nodal signal production in that community of ectodermal cells11,15. “Feed-back sub-circuits”, in which a downstream transcription factor feeds back to maintain activation of an upstream transcription factor, and “feed-forward sub-circuits”, in which an upstream transcription factor feeds forward to activate multiple sequential downstream steps, were found. Feed-back and feed-forward sub-circuits tended to stabilize regulatory states4 and contribute to the unidirectional trajectory of development. Sub-circuits just upstream of differentiation were found that drive the system forward and contribute to the activation of genes necessary for differentiation and cell biological function (e.g., 6,16–18). These and other sub-circuits provide dGRNs with modular design features that control explicit functions. As details of dGRN topology were identified, they were shown to contribute impressively to a growing understanding of developmental mechanisms in many systems and are central to research in sea urchin development as a consequence.\n\nWhen an early draft of the Strongylocentrotus purpuratus genome became available, it provided an opportunity to identify hundreds of transcription factor genes and molecules of signal transduction pathways (Figure 1). Each candidate development regulatory gene (transcription factors and signal transduction pathway gene) was assayed by quantitative polymerase chain reaction (qPCR) and whole-mount in situ hybridization (WMISH) to establish spatial and temporal expression patterns throughout early development19–25. Of the large number of transcription factors identified, those that were spatially or temporally distinct (or both) in expression in the early embryo were chosen for detailed efforts to define the distinct regulatory states of the embryo at a number of time points, beginning with fertilization and continuing through gastrulation. Ubiquitously expressed genes initially were excluded from the study with the notion that they were less likely to be involved in developmental regulatory decisions. Perturbation analyses of each identified signaling molecule and transcription factor then established linkages between nodes. Morpholino antisense oligonucleotides knocked down one transcription factor or signal, and analyses by qPCR, WMISH, or nanostring (or a combination of these) assayed the effect on other genes expressed at the same time, or in the same cell type, or both. This established a hierarchical relationship between the transcription factors, signals, and the genes encoding them in each cell type over time. The regulatory interactions were assembled into a network model by using BioTapestry as a platform for visualizing network topologies26,27. Many of the interactions were validated by using cis-regulatory analysis to determine direct binding of the transcription factor to specific sequences in the regulatory DNA of downstream genes. With this logical approach, hundreds of experiments were performed to establish sea urchin dGRN states in each emerging cell type up to gastrulation, and current efforts continue to extend the analysis to later time periods in development (e.g., 18,28–30).\n\nThe process begins with identification of candidate molecules. Candidates for the sea urchin dGRN were defined as transcription factors or signal transduction pathway members that were expressed in spatiotemporally specific patterns in the embryo. The regulatory linkages were established by conducting perturbation analyses in which one candidate was perturbed and asking how its loss affected expression of other candidates. These established a preliminary dGRN model. That model was challenged in many ways, including testing predictions that gene A activated gene B and gene C through cis-regulatory analysis.\n\n\nPatterning\n\nThe term “patterning” describes processes that establish the body plan of an organism. Patterning processes provide “positional information” enabling cells to know their location in an embryo. These processes direct morphogenesis, and they provide short-range and long-range signaling inputs to accomplish construction of a three-dimensional plant or animal (Figure 2). Each of these processes is driven by dGRNs. The sea urchin embryo begins patterning almost immediately after fertilization. By the 16-cell stage, zygotic transcription plus maternal inputs defines at least three distinct dGRN states (Figure 2). At this time, the future mesoderm and endoderm are combined as an endomesoderm network state. A Delta-Notch signal separates progeny of these cells into mesoderm (cells that receive the Delta signal) and endoderm (endomesoderm progeny that do not receive the Delta signal)31–33, and further specification plus signaling subdivides the initial endomesoderm GRN state and ectoderm GRN state until there are at least 15 different cell types recognizable by early gastrulation (Figure 2)7,34.\n\nOver the 24 hours from fertilization to the pluteus larval stage of Lytechinus variagatus, the number of developmental gene regulatory network (dGRN) states increases until there are more than 15 cell types in the early larva.\n\nSignaling is integral for the patterning mechanisms that organize multicellular cell and tissue types. As specification progresses, signaling establishes the three axes of the embryo. Early patterning signals divide the embryo into regions along the animal-vegetal axis, established by asymmetrically localized maternal information, followed by a series of Wnt signaling events that progressively diversify cell identities along the anterior-posterior axis35–41. The anterior-posterior axis of the larva is approximately identical to the animal-vegetal axis of the egg. At about mid-cleavage, the dorsal-ventral (D-V) axis is specified through Nodal and bone morphogenetic protein (BMP) signaling15,42,43, and toward the end of gastrulation, the right-left axis is established, again using Nodal and BMP and adding Hedgehog as a contributing signal16,44–47. Thus, by the end of gastrulation, signaling inputs plus a progression of transcription factor activations establish dGRN states for each cell type in the embryo, and even within the same germ layer, patterning inputs provide localized cell identities.\n\nPatterning continues to play a key role in organizing structures in the larva. As an example, a biomineralized skeleton provides the shape to the pluteus larva. To pattern that skeleton, signals from the ectoderm are received by the mesodermal skeletogenic cells, enabling them to position themselves correctly and to synthesize the calcium carbonate biomineral in the correct pattern48–51. Ectodermal signals are supplied from specific locations requiring the ectoderm to be patterned in advance. Ectodermal positional information leads to specification of two lateral patches of ectodermal cells specialized in secretion of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF)5,6,8,49,50. To provide that positional information, orthogonal bands of ectoderm are specified by even earlier signals. One of the ectodermal bands, the ciliary band, is specified at the boundary between the dorsal and ventral ectoderm as a consequence of Nodal (ventral) and BMP (dorsal) signaling15,42,52,53. BMP is synthesized in the ventral ectoderm and is transported, probably using Chordin as a chaperone, to induce dorsal ectoderm specification29,53,54. The ciliary band then forms between the dorsal and ventral ectodermal territories and is further subdivided29. The other ectodermal band, referred to as the border ectoderm, is specified in posterior ectoderm, just above the endoderm and orthogonal to the ciliary band separating the D-V ectoderm compartments. Wnt5 signaling from the endoderm is reported to induce the border ectoderm band in Lytechinus55,56, although a different Wnt is thought to be responsible in S. purpuratus56, and that band also receives input from the Nodal-BMP signaling as seen in both Lytechinus and Paracentrotus30,55. The site where the ciliary band and the border ectoderm band intersect becomes the signaling center that produces VEGF and FGF, both of which are necessary for initiation and growth of the skeleton49–51. Other signals also provide patterning inputs into skeletogenesis58. These data demonstrate that, in an organism, cells can be specified independently for long periods of time to seemingly establish independent dGRNs but that, at a later time, through signaling their dGRNs functionally intertwine once again. A good example is skeletal patterning, where signals from specific locations in the ectoderm provide patterning information and growth factors that direct the behavior of the mesoderm.\n\n\nMorphogenetic sub-circuits\n\nMorphogenetic control circuitry is another area where the dGRN is valuable (Figure 3). Each morphogenetic change incorporates multiple cell biological functions: changes in adhesion, motility, directionality, polarity, and so on. Perturbations that assess which transcription factors control individual cell’s biological properties have been valuable in dissecting details of those processes17,18,51,56,59–62. In an epithelial-mesenchymal transition (EMT), for example, five different dGRN sub-circuits control de-adhesion, motility, cell polarity, cell shape change, and invasion components of the EMT process61.\n\nMaternal inputs initiate specification. As cells divide, signaling becomes increasingly used to shape the specification sequence of each cell type. Toward the end of that process and proximal to differentiation, transcription factor sub-circuits drive expression of genes involved in differentiation and in morphogenesis.\n\nLater in development, a specific feed-forward sub-circuit controls the directed migration of the presumptive primordial germ cells18. Identification of such sub-circuits can then be used to penetrate the cell biology of each component process for each of the cell types (or organs) that have been defined in the larva. Along these lines, the circuits controlling ciliary band specification were identified, allowing for further analysis of the substrate for neural differentiation29. Yet, other circuits that control myogenesis of the larva were recently identified28. In each of these cases, the dGRN was valuable in gaining access to differentiation genes and effector genes ultimately controlled by that developmental GRN (Figure 3).\n\n\nEvolution\n\nA third valuable reason for studying dGRNs is to learn how evolutionary processes have changed dGRNs during species diversification. The sea urchin dGRNs are modeled primarily from data generated from three species of euechinoid sea urchins that are separated from one another by about 50 million years or less63. Examination of more distantly related species reveals how rewiring of dGRNs correlates with evolutionary changes in morphology and cell behavior. For example, an examination of a cidaroid sea urchin, separated from the euechinoids by more than 255 million years, revealed significant rewiring of the dGRN that specifies the larval skeletogenic lineage64,65. In euechinoids such as S. purpuratus or Lytechinus variagatus, the skeletogenic lineage is specified early during cleavage stages, and after a species-specific number of cell divisions, all of the skeletogenic cells undergo EMT before gastrulation of the other mesodermal and endodermal lineages. In the cidaroid, Eucidaris, the number of skeletogenic cells varies from embryo to embryo, and the EMT of these skeletogenic cells does not occur until much later in development relative to Lytechinus66. In Strongylocentrotus and Lytechinus, the skeletogenic lineage is specified by a core set of genes, including Alx1, Tbr, and Ets1, which are transcriptionally activated specifically in this lineage by unlocking a double-negative derepression sub-circuit controlled by Pmar1 and HesC4. In the cidaroid, Eucidaris, this double-negative derepression sub-circuit appears to be completely missing, and the spatiotemporal expression patterns of Alx1, Tbr, and Ets1 are different as well64. Thus, extensive rewiring of the dGRN occurs in sketogenic cell lineage, and morphogenesis of the skeletogenic cells differs, yet both modern sea urchins and the pencil urchins produce similar larval skeletons.\n\nComparative dGRN analysis can also reveal what aspects of specification are highly conserved during evolution. For example, experiments perturbing the function of Alx1 in euechinoids, cideroids, and sea cucumber have shown that Alx1 has a conserved role in promoting larval skeletogenesis64,67. At even larger evolutionary distance within echinoderms, valuable insights have been obtained through comparisons of skeletal, gut, and neural specification in sea stars compared with sea urchins. In each case, aspects of central dGRN circuitry were very similar despite about 500 million years of separation from the common ancestor68–70. dGRNs can also be used as a tool for understanding how circuitry has been co-opted during evolution. In a recent study, for example, it was learned that a conserved feed-forward sub-circuit involving Pax6, Six3, Six1/2, Eya, and Dach1 controls expression of the signal necessary for homing of primordial germ cells to the future gonad18. That circuit is very similar to the feed-forward circuit that controls specification of the Drosophila eye and vertebrate muscle, suggesting that such circuits may in some cases evolve as units of function, in this case by providing a feed-forward device for directed cell migration.\n\n\nConclusions\n\nSea urchin dGRNs describe the sequence of specification of all cells in the embryo up to the end of gastrulation. dGRN topology models produced in BioTapestry (http://sugp.caltech.edu/endomes/) record the current status of the network in S. purpuratus. Much more than a graphic description, it reflects a large number of experiments, where each connection is supported by multiple tests of the hypothesis that the expression of gene A activates or represses gene B. In its current form, the sea urchin dGRN includes more than 100 transcription factors and a number of signaling pathways, and in most cases multiple laboratories have validated each connection in S. purpuratus, and most are the same in Lytechinus and Paracentrotus.\n\nWith a high level of confidence in the structure of the sea urchin dGRNs, they became useful as a tool for exploring many other developmental questions. Here, we describe how the dGRNs have been used to inform patterning mechanisms, especially those necessary to produce the larval skeleton. We show how they have been useful in gaining a greater understanding of an EMT and a directed cell movement mechanism, both components of morphogenesis. We also describe how dGRNs are used as tools for discovery of evolutionary relationships.\n\nThe growth in our understanding of dGRNs has provided ways to address many unanswered questions. The ability to trace the entire specification trajectory of a cell type until it terminally differentiates is now a realistic goal. That ability has enormous power because it allows one to interrogate, dissect, and understand how that cell type arises and how it works in detail. This will be valuable in understanding the entire history of neurogenesis, for example, and in determining the mechanisms by which neurons diversify toward different neurotransmitter cell types. Other cell types can be followed with the same goal. dGRNs will also help us to understand how the upstream circuitry controls other morphogenetic movements in the early embryo. Thus, the information in the dGRN is useful both for gaining an intrinsic understanding of how developmental control circuitry works and as a tool for understanding patterning, morphogenesis, and evolutionary change.", "appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nSupport for this article was provided by grants from the National Institutes of Health (RO1-HD14483, PO1-HD03705 to DRM).\n\n\nReferences\n\nDavidson EH, Rast JP, Oliveri P, et al.: A genomic regulatory network for development. Science. 2002; 295(5560): 1669–78. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nSea Urchin Genome Sequencing Consortium, Sodergren E, Weinstock GM, et al.: The genome of the sea urchin Strongylocentrotus purpuratus. Science. 2006; 314(5801): 941–52. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSethi AJ, Wikramanayake RM, Angerer RC, et al.: Sequential signaling crosstalk regulates endomesoderm segregation in sea urchin embryos. Science. 2012; 335(6068): 590–3. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nRange RC, Angerer RC, Angerer LM: Integration of canonical and noncanonical Wnt signaling pathways patterns the neuroectoderm along the anterior-posterior axis of sea urchin embryos. PLoS Biol. 2013; 11(1): e1001467. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nDuboc V, Lapraz F, Saudemont A, et al.: Nodal and BMP2/4 pattern the mesoderm and endoderm during development of the sea urchin embryo. Development. 2010; 137(2): 223–35. PubMed Abstract \| Publisher Full Text\n\nHaillot E, Molina MD, Lapraz F, et al.: The Maternal Maverick/GDF15-like TGF-β Ligand Panda Directs Dorsal-Ventral Axis Formation by Restricting Nodal Expression in the Sea Urchin Embryo. PLoS Biol. 2015; 13(9): e1002247. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nDuboc V, Röttinger E, Lapraz F, et al.: Left-right asymmetry in the sea urchin embryo is regulated by nodal signaling on the right side. Dev Cell. 2005; 9(1): 147–58. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nWalton KD, Warner J, Hertzler PH, et al.: Hedgehog signaling patterns mesoderm in the sea urchin. Dev Biol. 2009; 331(1): 26–37. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBessodes N, Haillot E, Duboc V, et al.: Reciprocal signaling between the ectoderm and a mesendodermal left-right organizer directs left-right determination in the sea urchin embryo. PLoS Genet. 2012; 8(12): e1003121. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMaterna SC, Swartz SZ, Smith J: Notch and Nodal control forkhead factor expression in the specification of multipotent progenitors in sea urchin. Development. 2013; 140(8): 1796–806. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nArmstrong N, Hardin J, McClay DR: Cell-cell interactions regulate skeleton formation in the sea urchin embryo. Development. 1993; 119(3): 833–40. PubMed Abstract\n\nDuloquin L, Lhomond G, Gache C: Localized VEGF signaling from ectoderm to mesenchyme cells controls morphogenesis of the sea urchin embryo skeleton. Development. 2007; 134(12): 2293–302. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nRöttinger E, Saudemont A, Duboc V, et al.: FGF signals guide migration of mesenchymal cells, control skeletal morphogenesis [corrected] and regulate gastrulation during sea urchin development. Development. 2008; 135(2): 353–65. PubMed Abstract \| Publisher Full Text\n\nAdomako-Ankomah A, Ettensohn CA: Growth factor-mediated mesodermal cell guidance and skeletogenesis during sea urchin gastrulation. Development. 2013; 140(20): 4214–25. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nDuboc V, Lapraz F, Besnardeau L, et al.: Lefty acts as an essential modulator of Nodal activity during sea urchin oral-aboral axis formation. Dev Biol. 2008; 320(1): 49–59. PubMed Abstract \| Publisher Full Text\n\nLapraz F, Besnardeau L, Lepage T: Patterning of the dorsal-ventral axis in echinoderms: insights into the evolution of the BMP-chordin signaling network. PLoS Biol. 2009; 7(11): e1000248. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nBradham CA, Oikonomou C, Kühn A, et al.: Chordin is required for neural but not axial development in sea urchin embryos. Dev Biol. 2009; 328(2): 221–33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcIntyre DC, Seay NW, Croce JC, et al.: Short-range Wnt5 signaling initiates specification of sea urchin posterior ectoderm. Development. 2013; 140(24): 4881–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcIntyre DC, Lyons DC, Martik M, et al.: Branching out: origins of the sea urchin larval skeleton in development and evolution. Genesis. 2014; 52(3): 173–85. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCui M, Siriwon N, Li E, et al.: Specific functions of the Wnt signaling system in gene regulatory networks throughout the early sea urchin embryo. Proc Natl Acad Sci U S A. 2014; 111(47): E5029–38. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nPiacentino ML, Ramachandran J, Bradham CA: Late Alk4/5/7 signaling is required for anterior skeletal patterning in sea urchin embryos. Development. 2015; 142(5): 943–52. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nEttensohn CA: Lessons from a gene regulatory network: echinoderm skeletogenesis provides insights into evolution, plasticity and morphogenesis. Development. 2009; 136(1): 11–21. PubMed Abstract \| Publisher Full Text\n\nEttensohn CA: Encoding anatomy: developmental gene regulatory networks and morphogenesis. Genesis. 2013; 51(6): 383–409. PubMed Abstract \| Publisher Full Text\n\nSaunders LR, McClay DR: Sub-circuits of a gene regulatory network control a developmental epithelial-mesenchymal transition. Development. 2014; 141(7): 1503–13. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nRafiq K, Shashikant T, McManus CJ, et al.: Genome-wide analysis of the skeletogenic gene regulatory network of sea urchins. Development. 2014; 141(4): 950–61. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nMcClay DR: Evolutionary crossroads in developmental biology: sea urchins. Development. 2011; 138(13): 2639–48. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nErkenbrack EM, Davidson EH: Evolutionary rewiring of gene regulatory network linkages at divergence of the echinoid subclasses. Proc Natl Acad Sci U S A. 2015; 112(30): E4075–84. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nGao F, Thompson JR, Petsios E, et al.: Juvenile skeletogenesis in anciently diverged sea urchin clades. Dev Biol. 2015; 400(1): 148–58. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nWray GA, McClay DR: The origin of spicule-forming cells in a 'primitive' sea urchin (Eucidaris tribuloides) which appears to lack primary mesenchyme cells. Development. 1988; 103(2): 305–15. PubMed Abstract\n\nMcCauley BS, Wright EP, Exner C, et al.: Development of an embryonic skeletogenic mesenchyme lineage in a sea cucumber reveals the trajectory of change for the evolution of novel structures in echinoderms. Evodevo. 2012; 3(1): 17. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nHinman VF, Nguyen AT, Cameron RA, et al.: Developmental gene regulatory network architecture across 500 million years of echinoderm evolution. Proc Natl Acad Sci U S A. 2003; 100(23): 13356–61. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHinman VF, Nguyen A, Davidson EH: Caught in the evolutionary act: precise cis-regulatory basis of difference in the organization of gene networks of sea stars and sea urchins. Dev Biol. 2007; 312(2): 584–95. PubMed Abstract \| Publisher Full Text\n\nYankura KA, Martik ML, Jennings CK, et al.: Uncoupling of complex regulatory patterning during evolution of larval development in echinoderms. BMC Biol. 2010; 8: 143. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation" }	[ { "id": "12463", "date": "22 Feb 2016", "name": "Thierry Lepage", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12464", "date": "22 Feb 2016", "name": "Veronica Hinman", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12465", "date": "22 Feb 2016", "name": "Yi-Hsien Su", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-203
https://f1000research.com/articles/5-202/v1	22 Feb 16	{ "type": "Review", "title": "Current Landscape of Antiviral Drug Discovery", "authors": [ "Wade Blair", "Christopher Cox", "Christopher Cox" ], "abstract": "Continued discovery and development of new antiviral medications are paramount for global human health, particularly as new pathogens emerge and old ones evolve to evade current therapeutic agents. Great success has been achieved in developing effective therapies to suppress human immunodeficiency virus (HIV) and hepatitis B virus (HBV); however, the therapies are not curative and therefore current efforts in HIV and HBV drug discovery are directed toward longer-acting therapies and/or developing new mechanisms of action that could potentially lead to cure, or eradication, of the virus. Recently, exciting early clinical data have been reported for novel antivirals targeting respiratory syncytial virus (RSV) and influenza (flu). Preclinical data suggest that these new approaches may be effective in treating high-risk patients afflicted with serious RSV or flu infections. In this review, we highlight new directions in antiviral approaches for HIV, HBV, and acute respiratory virus infections.", "keywords": [ "Drug discovery", "heteroaryldihydropyrimidines", "phenylpropenamides", "F-protein", "monoclonal antibody", "influenza", "hepatitis B virus", "human immunodeficiency virus", "respiratory syncytial virus" ], "content": "Introduction\n\nViruses are intracellular pathogens that have evolved many devious strategies to evade host immune responses and, as a consequence, have plagued human health throughout history. Combating viral diseases with vaccines or antiviral drugs, or both, is a constant challenge. Even when successful strategies are discovered and employed, the high rate of genetic change exhibited by many viruses, particularly RNA viruses, often enables drug resistance or vaccine escape. This is compounded by the periodic emergence of new viral pathogens. Therefore, the continued search for new antiviral approaches is a noble cause that is critical for global human health.\n\nOver the last several decades, significant resources in academic and biotechnological/pharmaceutical research have been directed toward chronic viral infections such as HIV, HBV, and hepatitis C virus (HCV), resulting in breakthrough therapies that have had a major impact on these chronic diseases. In fact, HCV drug development represents one of the greatest success stories in the history of antiviral therapy. Antiviral medications have been recently developed that can achieve a sustained viral response (SVR) (virus eradication or immune control of virus replication in the absence of continued therapy) in a majority of HCV-infected patients after short treatment durations1. No less impressive has been the advent of combination antiretroviral therapy (cART) that has transformed HIV from a death sentence into a chronic but manageable condition. Similarly, some of the same approaches taken for HIV have been applied to HBV, resulting in the development of nucleoside/nucleotide analogues that effectively control viral replication and reduce the risk of HBV-associated disease, such as liver cirrhosis and hepatocellular carcinoma2. Although current therapies for HIV and HBV are effective for controlling viral replication, they do not achieve virus eradication or SVR, as is the case for HCV therapies. Therefore, HIV and HBV treatment strategies continue to evolve.\n\nTremendous efforts are currently being applied to eradication, or cure, of HIV3; however, this subject is beyond the scope of this review. In the absence of a viable cure for HIV, which even in the most optimistic view is still a decade away, the major unmet need in HIV treatment is to improve cART adherence. The prospect of lifelong therapy, combined with tolerability issues, leads to adherence challenges for many patients. In fact, more than 40% of patients with HIV experience some level of non-adherence over time on therapy4, and this can lead to incomplete suppression of viral replication, emergence of drug resistance, and, ultimately, therapeutic failure. To address this issue, long-acting antiretroviral agents (e.g. cabotegravir and rilpivirine) are currently being advanced in the clinic with the hope that less frequent, or supervised, dosing may improve adherence to antiretroviral therapy or enable wider use of preventative treatment. Cabotegravir and rilpivirine target HIV integrase and reverse transcriptase, respectively, and are currently being studied in phase 3 clinical trials as a two-drug combination for HIV maintenance therapy. The cabotegravir and rilpivirine regimen is administered once every 4 or 8 weeks as multiple intramuscular injections following full suppression on an oral regimen5. Recent data from the LATTE 2 trial showed that the injectable cabotegravir/rilpivirine combination maintained viral suppression rates comparable to a three-drug oral regimen of cabotegravir and two nucleoside reverse transcriptase inhibitors after 32 weeks6. Patient surveys have indicated enthusiasm for long-acting parental therapies in the HIV community7; however, it remains to be seen whether the reduction in dosing frequency and new administration route can improve adherence in certain patient populations. In addition, several questions remain about the potential liabilities of long-acting antiretrovirals, such as the risk of adverse events and drug resistance with prolonged exposure at subtherapeutic levels in patients who exhibit periodic lapses in adherence.\n\n\nHepatitis B virus\n\nHBV drug discovery efforts are currently geared toward increasing SVR rates, defined for HBV as HBsAg loss/seroconversion and control of HBV viral load in the absence of therapy. Current standard of care for chronically infected patients with HBV (CHB) is nucleoside/nucleotide analogue therapy (e.g. entecavir) or interferon (IFN). Nucleoside/nucleotide analogue therapy is highly effective at suppressing viral load, and a low percentage of treated CHB patients achieve SVR (<10%) after long-term treatment (2 to 4 years)2. Similar rates of IFN-treated patients achieve SVR (<10%) after 48 weeks of treatment; however, IFN therapy is not well tolerated. Identification of direct-acting antivirals targeting new mechanisms in the HBV replication cycle that could be combined with nucleoside/nucleotide analogues or IFN therapy (or both) to achieve increased SVR rates is one concept under current investigation.\n\nThe HBV capsid represents an emerging HBV target that is potentially interesting. Originally, two classes of HBV capsid inhibitors were described: the heteroaryldihydropyrimidines (HAPs) and the phenylpropenamides8. Mechanistic studies revealed that HAP compounds increase the kinetics of assembly and stabilize HBV capsid dimer interactions, resulting in aberrant capsid assembly9,10. Similar to HAPs, phenylpropenamides (e.g. AT-130) accelerate capsid assembly; however, this class of compounds also blocks RNA packaging, resulting in the formation of empty capsids rather than misdirecting capsid assembly11. BAY 41-409, a member of the HAP class, demonstrated antiviral activity in an HBV transgenic mouse model and in humanized mice infected with HBV12,13. The disclosure of an HBV capsid/inhibitor X-ray cocrystal showed that the two classes of inhibitors shared overlapping binding sites on the HBV capsid14. Recently, additional classes of HBV capsid inhibitors have been disclosed15,16, suggesting that the HBV capsid can be targeted by diverse chemical matter. In addition, capsid inhibitors with analogous activity have been identified for picornaviruses17, HIV8, and Dengue virus18, strongly suggesting that viral capsid proteins may represent a viable target for a broad range of viruses. However, proof of concept (POC) in the clinic remains to be demonstrated.\n\n\nRespiratory syncytial virus and flu\n\nIn addition to chronic viral infections, a great deal of preclinical research has recently been focused on direct-acting antivirals against negative-stranded RNA viruses that cause respiratory infections, most notably for RSV and influenza (flu). Despite many years of intensive research efforts, there are very limited treatment options for these viral diseases that take their toll on the most sensitive of patient populations: the very young, the very old, and the immunocompromised. The development of efficacious flu vaccines has reduced morbidity and mortality in patients aged 6 months to 65 years, but inability to treat infants and reduced efficacy in the elderly leave a significant gap in patient coverage. Additionally, in years where strain mismatch occurs (e.g. 2014), a significant increase in the number of flu-related hospitalizations and deaths is observed19. The currently available agents, which target either the neuraminidase enzyme or M2 protein, leave much to be desired in terms of efficacy, treatment window, and resistance profile. Our defense against RSV is even more sparse; there is no vaccine available, and ribavirin and palivizumab—Synagis (MedImmune, Gaithersburg, MD, USA), a monoclonal antibody (mAb) directed against the RSV fusion protein—are the only licensed agents for RSV; use of the former has been significantly reduced because of concerns over efficacy and toxicity, and the latter is approved only for prophylactic use in infants at highest risk.\n\nThe majority of reported drug discovery efforts against RSV over the past two decades have been focused on the F- (or fusion) protein, with multiple antibodies and small molecules, as well as a nanobody, entering development20; some of these agents have been halted for efficacy, safety, or strategic reasons, but several are progressing through the clinic as highlighted below. An X-ray crystal structure of the post-fusion F-protein became available in 201121, and it remains to be seen whether the structural insights gained have encouraged additional directed efforts at this validated target. Several other viral proteins, including the N-protein and the G-protein, have been targeted by antibodies, small molecules, and the first antiviral small interfering RNA (siRNA)20. Inhibition of the L-protein, the RNA-dependent RNA-polymerase of RSV, seems an ideal target given its critical function in viral replication; however, little success has been realized prior to the 2014 disclosure of the leading L-protein inhibitor AL-8176 (vide infra). The recent disclosure of an RSV replicon assay22 and a screening cascade to identify RSV inhibitors in a target-agnostic fashion23 demonstrate the continued desire both in academia and in the pharmaceutical industry to discover novel mechanisms of action for the treatment of RSV.\n\nIn terms of small-molecule development, 2014 was a watershed year that witnessed the first human POC for two mechanisms of action: the fusion inhibitor GS-5806 (Gilead, Foster City, CA, USA)24 and the nucleoside inhibitor of the L-protein, AL-8176 (Alios, South San Francisco, CA, USA)25 both demonstrated efficacy in human challenge studies26,27. The compounds were well tolerated, reduced symptoms associated with disease, and dramatically decreased viral load; the Alios nucleoside reduced viral burden in nasal washes to undetectable levels, whereas the fusion inhibitor produced a 4-log drop in viral titer. One is tempted to speculate that targeting the L-protein may lead to better efficacy (as suggested by undetectable viral load in the challenge study) and a higher barrier to resistance than targeting surface interactions; additionally, according to Alios pipeline reports, AL-8176 is also being advanced for additional paramyxovirus infections of medical concern: parainfluenza virus and human metapneumovirus28. An agent such as this with broad-spectrum antiviral activity could be a true game-changer in the treatment of respiratory virus infections in high-risk populations. Other agents currently in clinical trials include second-generation (more potent and half-life extended) mAb against F-protein, MEDI-8897, (MedImmune); REGN-2222, another anti-RSV F mAb, (Regeneron, Tarrytown, NY, USA); an inhaled nanobody from Ablynx (Ghent/Zwijnaarde, Belgium), ALX-0171, directed against F-protein; and the inhaled siRNA from Alnylam (Cambridge, MA, USA), ALN-RSV01, that targets a conserved epitope on N-protein. Additionally, the F-protein inhibitor AK0529, from Ark Biosciences (Shanghai, China), recently completed phase 1 studies and is now undergoing phase 2 evaluation in hospitalized infants29. Given the high unmet medical need and several tractable, validated mechanisms to target, we expect to see a continued high level of competition in the RSV space and are confident that novel agents will begin to arrive on the market in the near future.\n\nFlu research has witnessed a recent surge in activity, and most efforts are focused on novel mechanisms of action that may have potential for improved efficacy, treatment window, and resistance profile when compared with neuraminidase or M2 protein inhibitors. To this end, we feel that the brightest future lies in inhibition of one or more components of the viral polymerase complex. Unlike most negative-stranded RNA viruses, Orthomyxoviruses such as flu use a unique heterotrimeric polymerase complex composed of the PA protein (endonuclease), the PB1 protein (the RNA-dependent RNA-polymerase, or RdRp), and the PB2 protein (cap-snatching subunit) that work together in a tightly associated and coupled fashion30. The past decade has seen significant advances in our understanding of the structure and function of the subunits of the polymerase complex, culminating in the recent elucidation of the full heterotrimeric polymerase complex of flu A31, flu B32, and flu C33 by X-ray crystallography. As is the case for targeting RSV polymerase, the flu polymerase complex is a compelling target for novel antivirals because of its role in not just reducing the spread of infection but also halting all intracellular replication; this may lead to an expanded window for intervention and reduced likelihood for the generation of drug-resistant viruses. Compounds targeting each of the three components of the polymerase have now reached the stage of clinical evaluation.\n\nThe most advanced compound is the PB1 inhibitor Favipiravir (T-705), which is approved for use against pandemic flu in Japan; additional clinical studies are ongoing around the world to study its safety and efficacy in the treatment of uncomplicated flu. The pyrazinecarboxamide of T-705 is converted to a nucleoside analog in vivo and is believed to act via incorporation into viral RNA by the PB1 RdRp; however, as with ribavirin, it is a non-specific nucleoside that is active against multiple viruses and has been shown to induce lethal mutagenesis in flu strains in vitro34. Favipiravir has also shown efficacy for Ebola infection in mouse models of disease35,36 and was administered post-infection during the recent Ebola outbreak, where it showed some evidence of efficacy if administered prior to significant disease onset37. Development of nucleosides that specifically target the PB1 protein has been a substantial challenge in the field; however, a recent patent application from Riboscience (Palo Alto, CA, USA) suggests that potent and specific nucleosides for this purpose may be achievable38. The current stage of development of these nucleoside analogs is unknown.\n\nThe most exciting development of the past several years has been identification of the first inhibitor of the cap-snatching function of the PB2 protein via a phenotypic screening approach39. In 2013, Vertex (Boston, MA, USA) achieved POC in a human challenge study with their leading PB2 inhibitor, VX-787, wherein they demonstrated significant reductions in viral load and symptom duration when drug was administered, beginning 24 hours after exposure to the virus, once a day for 5 days40. Preclinical data from a lethal mouse flu model indicate that VX-787 does indeed expand the treatment window to 96 hours post-infection; in contrast, oseltamivir must be given within 48 hours to provide a measure of protection in this model41; however, delayed treatment efficacy has not yet been tested in humans. In June 2014, Janssen (Beerse, Belgium) entered into a collaborative agreement with Vertex to develop and commercialize the renamed compound (JNJ-872). JNJ-872 has broad-spectrum activity against flu A strains but does not have activity against flu B. This observation can be readily explained by examination of the X-ray structure of JNJ-872 bound to the flu A PB2 protein, wherein many of the amino acids in direct contact with the inhibitor are not conserved between the major flu A and B strains41. These structural data suggest that identification of a second-generation inhibitor that provides efficacy against both flu A and B will be very challenging to obtain within a similar binding motif. Roche (Basel, Switzerland) and a small biotech, Savira (Vienna, Austria), founded by the group that solved the full flu polymerase structures, partnered in 2013 to develop flu polymerase inhibitors, but their current stage of development is unknown.\n\nThe endonuclease function of the PA protein is the subunit with the longest history; the first inhibitors were reported by Merck (Kenilworth, NJ, USA) in the mid-1990s42. This enzyme uses a similar active site as HIV integrase; in fact, it was these early endonuclease inhibitors that led Merck to identify, via high-throughput screening of their sample collection, the key pharmacophore that has supplied all of the chemical matter advanced to date for the integrase enzyme, including the three US Food and Drug Administration-approved integrase inhibitors. The challenging pharmaceutical properties of small molecules required to inhibit endonuclease has likely slowed progress, but advances in HIV integrase compound design, as well as X-ray elucidation of the PA subunit in complex with inhibitors43,44, may have informed researchers how to make more drug-like endonuclease inhibitors. In fact, two companies have endonuclease inhibitors in clinical stage evaluation: Shionogi (Osaka, Japan) has demonstrated safety and acceptable pharmacokinetics of S-033188 in a phase 1 study in Japan45, and Janssen (through their acquisition of AL-794 from Alios) has an endonuclease inhibitor currently in phase 1 clinical studies in healthy volunteers in the US46. Though there is not yet POC for this mechanism, endonuclease’s vital function in the polymerase complex makes its clinical success highly probable. Furthermore, there is very high homology between flu A and B in the endonuclease active site, suggesting that this could be a better target than the currently identified site on PB2 to obtain broad-spectrum flu activity. If Janssen’s endonuclease compound AL-794 advances, they could be in a position to test combinations with JNJ-872 as early as 2017, providing the first look at the potential synergy in combining the inhibition of two separate components of the polymerase complex.\n\nUtilization of broadly neutralizing mAbs targeting the Flu hemagglutinin (HA) protein represents a separate approach to develop flu therapeutics. With advances in technologies to isolate antibodies directly from human B cells, many anti-flu HA mAbs with broad neutralizing activity have been identified47–54. Several such mAbs—FI6, CR9114, and 39.29 (MHAA4549A)—that target the highly conserved stem region of HA demonstrate potent activity against all, or nearly all, flu A strains tested51,53,54. One potential indication of interest is the utilization of flu mAbs to treat patients hospitalized with severe flu infection. Preclinical animal models indicate that flu mAbs exhibit superior efficacy compared with oseltamivir in mouse or ferret models of lethal flu infection when treatment is initiated later in infection54. Multiple mechanisms of antiviral activity have been described for flu mAbs, including Fc effector function-mediated activity55, providing some rationale for the improved activity observed in preclinical models. MHAA4549A is currently being developed in the clinic and has demonstrated POC in a human challenge study; however, the dose required for efficacy was high (3.6 grams). Despite the high dose requirements, MHAA4549A is progressing to phase 2 trials in hospitalized patients and will test the utility of HA stem-binding mAbs in this at-risk population. A second HA stem-binding mAb currently being developed by Visterra (Cambridge, MA, USA), VIS410, also recently demonstrated POC in a human challenge study at a similar high dose (2.3 grams)56.\n\n\nConclusion\n\nThough some pharmaceutical companies have recently announced a reduction or elimination of their efforts toward antiviral drug discovery, the future of research in this area remains bright; several large companies, as well as many biotechnology companies and academic researchers, are pushing the frontiers of knowledge and unearthing new approaches to combating viral infections. Along with the exciting future in new treatments for HIV, HBV, RSV, and flu highlighted above, nascent reports suggest that tractable approaches to treat viral diseases such as norovirus, Dengue, and Ebola are also within reach. Furthermore, the largely unexplored and poorly understood area of targeting the host immune system and harnessing its power to clear viral infections is gaining traction, and we are hopeful that such an approach may one day allow the discovery of heretofore unknown single agents that have the potential to cure diseases caused by a broad range of genetically distinct viruses57.\n\n\nAbbreviations\n\ncART, combination antiretroviral therapy; CHB, chronic hepatitis B; flu, influenza; HA, hemagglutinin; HAP, heteroaryldihydropyrimidine; HBV, hepatitis B virus; HCV, hepatitis C virus; HIV, human immunodeficiency virus; IFN, interferon; mAb, monoclonal antibody; POC, proof of concept; RdRp, RNA-dependent RNA-polymerase; RSV, respiratory syncytial virus; siRNA, small interfering RNA; SVR, sustained virological response.", "appendix": "Competing interests\n\n\n\nThe authors declare that they 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\nErmis F, Senocak Tasci E: New treatment strategies for hepatitis C infection. 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PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nhttps://www.viivhealthcare.com/media/press-releases/2015/november/viiv-healthcare-announces-positive-headline-results-from-a-study-of-two-drug-injectable-regimen-for-hiv-maintenance-therapy.aspx.\n\nWilliams J, Sayles HR, Meza JL, et al.: Long-acting parenteral nanoformulated antiretroviral therapy: interest and attitudes of HIV-infected patients. Nanomedicine (Lond). 2013; 8(11): 1807–13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKlumpp K, Crépin T: Capsid proteins of enveloped viruses as antiviral drug targets. Curr Opin Virol. 2014; 5: 63–71. PubMed Abstract \| Publisher Full Text\n\nBourne CR, Finn MG, Zlotnick A: Global structural changes in hepatitis B virus capsids induced by the assembly effector HAP1. J Virol. 2006; 80(22): 11055–61. 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PubMed Abstract \| Publisher Full Text\n\nWang G, Deval J, Hong J, et al.: Discovery of 4'-chloromethyl-2'-deoxy-3',5'-di-O-isobutyryl-2'-fluorocytidine (ALS-8176), a first-in-class RSV polymerase inhibitor for treatment of human respiratory syncytial virus infection. J Med Chem. 2015; 58(4): 1862–78. PubMed Abstract \| Publisher Full Text\n\nDeVincenzo JP, Whitley RJ, Mackman RL, et al.: Oral GS-5806 activity in a respiratory syncytial virus challenge study. N Engl J Med. 2014; 371(8): 711–22. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nDeVincenzo JP, McClure MW, Symons JA, et al.: Activity of Oral ALS-008176 in a Respiratory Syncytial Virus Challenge Study. N Engl J Med. 2015; 373(21): 2048–58. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nhttp://www.aliosbiopharma.com/pipeline/overview/.\n\nhttps://clinicaltrials.gov/ct2/show/NCT02460016.\n\nFodor E: The RNA polymerase of influenza a virus: mechanisms of viral transcription and replication. 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PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nOestereich L, Lüdtke A, Wurr S, et al.: Successful treatment of advanced Ebola virus infection with T-705 (favipiravir) in a small animal model. Antiviral Res. 2014; 105: 17–21. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nSmither SJ, Eastaugh LS, Steward JA, et al.: Post-exposure efficacy of oral T-705 (Favipiravir) against inhalational Ebola virus infection in a mouse model. Antiviral Res. 2014; 104: 153–5. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nhttp://www.msf.org/article/preliminary-results-jiki-clinical-trial-test-efficacy-favipiravir-reducing-mortality.\n\nSmith M, Klumpp KG: 4’-Difluoromethyl Substituted Nucleoside Derivatives as Inhibitors of Influenza RNA Replication. WO 2015/120237A2. 2015. Reference Source\n\nClark MP, Ledeboer MW, Davies I, et al.: Discovery of a novel, first-in-class, orally bioavailable azaindole inhibitor (VX-787) of influenza PB2. J Med Chem. 2014; 57(15): 6668–78. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nhttp://investors.vrtx.com/releasedetail.cfm?releaseid=744857.\n\nByrn RA, Jones SM, Bennett HB, et al.: Preclinical activity of VX-787, a first-in-class, orally bioavailable inhibitor of the influenza virus polymerase PB2 subunit. Antimicrob Agents Chemother. 2015; 59(3): 1569–82. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nTomassini J, Selnick H, Davies ME, et al.: Inhibition of cap (m7GpppXm)-dependent endonuclease of influenza virus by 4-substituted 2,4-dioxobutanoic acid compounds. Antimicrob Agents Chemother. 1994; 38(12): 2827–37. 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PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nEkiert DC, Bhabha G, Elsliger MA, et al.: Antibody recognition of a highly conserved influenza virus epitope. Science. 2009; 324(5924): 246–51. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nEkiert DC, Friesen RH, Bhabha G, et al.: A highly conserved neutralizing epitope on group 2 influenza A viruses. Science. 2011; 333(6044): 843–50. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSui J, Hwang WC, Perez S, et al.: Structural and functional bases for broad-spectrum neutralization of avian and human influenza A viruses. Nat Struct Mol Biol. 2009; 16(3): 265–73. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nCorti D, Voss J, Gamblin SJ, et al.: A neutralizing antibody selected from plasma cells that binds to group 1 and group 2 influenza A hemagglutinins. Science. 2011; 333(6044): 850–6. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nWrammert J, Koutsonanos D, Li GM, et al.: Broadly cross-reactive antibodies dominate the human B cell response against 2009 pandemic H1N1 influenza virus infection. J Exp Med. 2011; 208(1): 181–93. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nDreyfus C, Laursen NS, Kwaks T, et al.: Highly conserved protective epitopes on influenza B viruses. Science. 2012; 337(6100): 1343–8. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nNakamura G, Chai N, Park S, et al.: An in vivo human-plasmablast enrichment technique allows rapid identification of therapeutic influenza A antibodies. Cell Host Microbe. 2013; 14(1): 93–103. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nDiLillo DJ, Tan GS, Palese P, et al.: Broadly neutralizing hemagglutinin stalk-specific antibodies require FcγR interactions for protection against influenza virus in vivo. 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PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation" }	[ { "id": "12461", "date": "22 Feb 2016", "name": "Steven Projan", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12462", "date": "22 Feb 2016", "name": "David Margolis", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-202
https://f1000research.com/articles/5-201/v1	22 Feb 16	{ "type": "Review", "title": "Particulate air pollution and impaired lung function", "authors": [ "Laura Paulin", "Nadia Hansel", "Nadia Hansel" ], "abstract": "Air pollution is a leading cause of morbidity and mortality throughout the world, particularly in individuals with existing lung disease. Of the most common air pollutants, particulate matter (PM) is associated with an increased risk of exacerbations and respiratory symptoms in individuals with existing lung disease, and to a lesser extent, in those without known respiratory issues. The majority of published research has focused on the effects of PM exposures on symptoms and health care utilization. Fewer studies focus on the impact of PM on objective measurements of pulmonary function. This review will focus on the effects of PM exposure on objective measurements of lung function in both healthy individuals and those with existing lung disease.", "keywords": [ "Air pollution", "Particulate matter", "Lung function", "asthma", "COPD", "lung development" ], "content": "Introduction\n\nAir pollution is associated with millions of premature deaths worldwide1, 25% of which are estimated to be respiratory in nature2, and is the world’s largest environmental health risk3. In an attempt to mitigate ambient exposure to air pollution, the Environmental Protection Agency (EPA) currently regulates six criteria air pollutants under the Clean Air Act4. Of the regulated pollutants, particulate matter (PM) has been extensively studied and associated with a myriad of adverse health outcomes, including an adverse impact on lung function in both children and adults5–7. PM is a complex mixture of liquid droplets and extremely small particles composed of organic and inorganic compounds7. PM less than 10 μg in aerodynamic diameter, designated by PM10, can penetrate conducting airways; PM2.5 (PM less than 2.5 μg in aerodynamic diameter) is composed of fine particles and can penetrate into the gas-exchanging regions of the lung6. Sources of ambient PM include construction sites, smokestacks, fires, power plants, and automobiles; the main sources of indoor PM include ambient PM, tobacco smoke, cooking, and heating appliances. PM causes lung inflammation and mucous secretion by acting on airway epithelial cells and alveolar macrophages and may lead to airway remodeling8.\n\nObstructive lung diseases, including chronic obstructive pulmonary disease (COPD) and asthma, are a large source of morbidity and mortality across the entire world9,10. Exposure to PM has been associated with worse morbidity and mortality in patients with COPD and asthma, including worse symptoms and quality of life and higher exacerbation rates and health care utilization11–17. Lung function is often used as an indicator of disease severity, and several studies have also focused on the impact of PM exposure on objective measures of lung function. This review will focus on the health effects of PM exposure on lung function in healthy children and adults, and in those with existing pulmonary disease.\n\n\nImpact of PM exposure in utero and during infancy\n\nChildren may be particularly sensitive to the health effects of PM exposure, and early exposure during critical time points in lung development may lead to lasting impacts on lung function18–20. Several studies show that exposure to PM in utero is associated with decreased lung function in children, suggesting that fetal lung growth is impacted by maternal PM exposures21. For example, in a Californian study of 232 children with asthma, fetal exposure to ambient PM10 during the first trimester of pregnancy was associated with a lower peak expiratory flow (PEF) between the ages of 6 and 1122. In a study of 176 children of non-smoking mothers, Jedrychowski et al. measured maternal exposure to PM using a backpack worn for 48 hours and showed that children exposed to higher concentrations of PM2.5 in utero had an approximately 90 mL lower forced expiratory volume in 1 second (FEV1) at 5 years of follow-up23. Exposure to higher concentrations of PM10 during the first year of life was associated with a FEV1 reduction of nearly 60 mL in Swedish children aged 8 years old, suggesting that exposures during infancy have lasting health effects years later24. Importantly, analysis of the same cohort at 16 years of age shows that children with exposure to higher concentrations of PM10 during the first year of life (above the median concentration) were more likely to have FEV1 and forced vital capacity (FVC) less than the lower limit of normal25. While these studies suggest in utero and infancy PM exposure leads to lower lung function, others have not supported the relationship between early PM exposure and lung function decrements. In a German study of 2226 children, investigators used comprehensive land-use regression modeling to estimate yearly average PM concentrations at the time of each participant’s birth. Early long-term PM exposure was not found to be associated with spirometric outcomes at 15 years of age26. In a Swiss study, maternal exposure to PM10 measured daily during pregnancy was not found to be associated with lung volumes in 241 healthy newborns27. To address the knowledge gaps that persist, large cohort studies with frequent comprehensive participant evaluations and individual-level PM exposure assessments are necessary to identify potential time points of increased susceptibility of the developing lung.\n\n\nImpact of PM exposure in healthy children\n\nThe research evaluating the impact of PM exposure on lung function in healthy children is relatively scarce and inconsistent. Several observational studies demonstrate that PM exposure may have short- and long-term impacts on lung function in children without known lung disease. A longitudinal study by Castro et al. found that an increase in 2-day lagged PM was associated with a 0.34 L/min decrease in PEF in 118 schoolchildren in Rio de Janeiro28. Similarly, an air pollution episode resulting in a 1-day PM10 concentration of 174 μg/m3 was associated with worse same-day spirometry in 112 healthy Dutch children29. Higher concentration of 8-hour PM, measured on the morning prior to lung function testing, was associated with a marginal decrease in FEV1 and FVC in 163 healthy Austrian schoolchildren30. However, after accounting for nitrogen dioxide concentrations, this association was attenuated, highlighting the importance of identifying all exposures that may impact lung function.\n\nThe Children’s Health Study followed respiratory growth over a period of 8 years in 1759 Californian children. Children exposed to the highest PM2.5 concentrations had an approximately 80 mL lower FEV1 than children exposed to the lowest level of PM19, an effect size similar to observed values in children exposed to maternal smoking19,31,32. In the same cohort, children living in residences closer to roadways, with higher estimated PM concentrations, had approximately 3% lower FEV133, highlighting the contribution of traffic-related pollution to respiratory health. A study of 2307 healthy 10-year-old children in Norway found that an increase in the lifetime exposure to PM was associated with a 58–66 mL/sec lower PEF34, and in 179 British schoolchildren followed for several months, higher PM was associated with a 4% increase in the odds of having a lower PEF (defined as greater than 20% below the median)35.\n\nThere were no consistent associations between PM exposure and lung function in a 3-year longitudinal study of 1150 Austrian schoolchildren, including both healthy children and those with asthma36. In an analysis of data from the Six Cities Study, indoor measurements of PM2.5 showed no direct association with pulmonary function measurements in a random sample of 2994 children37. The discrepancy in research may be due to difficulty in performing lung function testing in children, or in part due to the nature of the exposure and unmeasured changes in PM concentration over time. Although there are few cohorts that extend from childhood to adulthood, these findings suggest that exposure in early life may have implications in the development of future airway disease and highlight the need for comprehensive longitudinal studies.\n\n\nImpact of PM exposure in children with asthma\n\nChildren with existing lung disease are likely to be more susceptible to lung function changes following exposure to PM. In a study of 326 elementary schoolchildren in Seattle, 24 of whom had asthma, a 20 μg/m3 increase in ambient PM2.5 concentration measured over 2 years at the children’s school was associated with a 34 and 37 mL drop in FEV1 and FVC, respectively, in those with asthma38. British children with wheezing had increased odds of having a drop in PEF when exposed to higher concentrations of outdoor PM measured over the course of a winter season35. Exposure to higher PM concentrations over shorter time periods can lead to changes in lung function as well. When exposed to higher concentrations of ambient PM2.5 over a 3-day period, children with asthma in Canada were found to have a decrease in FEV1 and FVC39. An increase of 10 μg/m3 in 24-hour mean PM was associated with a drop of 3 L/min in PEF in Japanese children hospitalized for severe asthma exacerbations40. Delfino et al. followed 53 children with asthma living in Los Angeles and found that an interquartile range (IQR) increase in 1-hour peak PM was associated with a 1.32% decrease in same-day percent predicted FEV141. In a study of 19 children with asthma, Allen et al. found that a higher 24-hour concentration of PM2.5, measured over a period of 10 days, was associated with worse lung function as measured by daily PEF and FEV142. Although less studied, short-term increases in indoor PM exposures have also been associated with lower lung function in children with asthma. A higher 24-hour indoor PM concentration measured over a period of 10 days was associated with a decrease in same-day and 1-day lagged PEF and FEV1 in Seattle children with asthma43. In another study, higher 24-hour indoor PM concentrations were associated with worse PEF in 22 children with asthma living in the United Kingdom44.\n\nStudies examining the relationship between PM exposure and lung function in children with asthma have been inconsistent. In the Children’s Health Study, there was no significant effect of PM exposure on lung function in the 483 children with a history of doctor-diagnosed asthma19. The authors suggest that the smaller sample size in this subgroup may have contributed to these findings. Noting the lack of consistency in quantitative outcomes, Weinmayr et al. completed a meta-analysis of 36 studies evaluating the association of PM and respiratory symptoms and PEF in children with asthma or asthma-like symptoms. Although there is clear evidence of worsening respiratory symptoms associated with higher PM concentrations, the authors report a non-significant decrease in PEF of 0.082 L/min per unit increase in PM45. Furthermore, investigators have often noticed a disconnect between asthma symptoms and lung function46, suggesting that lung function changes may not be the most sensitive marker of disease burden in children.\n\n\nImpact of PM exposure on adults\n\nAlthough the developing lung may be more vulnerable to PM exposures, several studies show that adults are susceptible to air pollution exposures as well. The SAPALDIA study of nearly 10,000 Swiss adults showed that a 10 μg/m3 increase in PM10 was associated with a 3.4% decrease in FVC in a cross-sectional analysis, even after adjusting for cigarette use47,48. In the Normative Aging Study, 858 elderly men living in the Boston area were followed for a period of over 10 years, and exposure to ambient black carbon (a marker of traffic-related PM) was estimated using spatiotemporal land use regression models. Higher yearly black carbon concentration was associated with an increase in the normal age-related rate of decline of FEV1 and FVC49. Similarly, higher concentrations of previous-day ambient PM2.5 were associated with approximately 20 mL lower FEV1 and FVC in nonsmoking adults in the Framingham Heart study50, and exposure to higher long-term concentrations of PM (yearly levels) was associated with lower FEV1 and a faster rate of lung function decline51. In this cohort, followed for over 10 years, a 10 μg/m3 increase in PM2.5 was associated with an additional 10.5 mL annual decline in FEV1 beyond the normal age-related decline, suggesting that exposure to even moderate concentrations of PM can lead to meaningful lung function decline in healthy adults. Though the absolute changes in lung function in the above studies are of relatively low magnitude, prior research has shown that decreases in FEV1 are associated with all-cause mortality52,53 and that lower lung function is associated with higher rates of cardiovascular disease54,55.\n\n\nAdults with existing lung disease\n\nThere are few studies investigating the effects of PM on lung function in adults with established lung disease, such as COPD and asthma. The studies that do exist suggest that among individuals with established COPD, long-term exposure to outdoor PM may be linked to lung function decline. For example, PM2.5 exposure was associated with FEV1 decline among 1218 subjects with severe COPD followed for an average of 29.2 months in the National Emphysema Treatment Trial (NETT)56. Similarly, among 401 individuals with COPD and α-1-antitrypsin deficiency, a 10 µg/m3 increase in PM10 was associated with an additional 30 mL/year decline in FEV157. Though these studies are suggestive, whether these findings extend to patients without α-1-antitrypsin deficiency or severe emphysema is unclear. Long-term studies of air pollution on a diverse group of subjects with COPD, including those with heavy smoking exposure, are needed to further clarify the effects of air pollution on COPD progression.\n\nShort-term variation in pollution may also be associated with changes in lung function in patients with chronic lung disease; however, studies investigating short-term variation in pollution and lung function in adults with COPD or asthma have been inconsistent and interpretations are limited by small sample sizes. For example, Peacock et al. recruited 94 COPD subjects who filled out daily diaries. An IQR increase in PM10 was linked to an approximately 13% increase in odds of symptomatic decreases in PEF, defined as a fall in PEF for at least 2 days plus a reported increase in dyspnea11. In a recent Italian study, increased outdoor PM10 concentrations were associated with lower FEV1 and FVC in COPD patients presenting with urgent hospitalization58. Similarly, Lagorio et al. showed that increased ambient PM2.5 and PM10 concentrations were associated with lower lung function (FEV1 and FVC) in subjects with COPD, and the effect on FEV1 appeared only when 72 hours of exposure were accumulated59. However, other studies have not shown a link between PM and short-term changes in lung function among those with COPD. A small panel study including 17 subjects with COPD showed no consistent association between PM with lung function over 12 days60. A cohort of patients with COPD followed for 3 months found that an increase in PM10 was associated with an increase in nighttime symptoms, but there was no change in lung function61. Similarly, Hansel et al. identified that indoor PM is linked to respiratory symptoms and exacerbations, but not lung function, among former smokers with COPD15.\n\nStudies that have investigated the short-term effects of PM on lung function in subjects with asthma have also been inconsistent. In a real-time exposure study of 60 adults with mild to moderate asthma, McCreanor et al. found that FEV1 decreased up to 6.1% following a 2-hour walk in a high-traffic area (with corresponding higher PM2.5 concentration) as compared to a walk in a park (with lower PM2.5 concentration) of similar duration62. In a longitudinal analysis, Park et al. found an increase in PM10 concentration was associated with increases in PEF variability of >20% and a decrease in the mean PEF among 64 adults with asthma63. Balmes et al. noted that those with asthma with the middle and highest tertiles of ambient PM2.5 exposure had an increased risk of having FEV1 below the lower limit of normal (odds ratio [OR]=1.93 95% confidence interval [CI]: 0.94, 3.95 and OR=2.23 95% CI: 1.08, 4.61), but only among females64. Using biweekly spirometry over 6 months on a group of 54 adult asthmatics, Penttinen et al. found that particle number concentrations on the preceding days were inversely, but mostly non-significantly, associated with FEV1, FVC, and PEF, and no associations were observed with larger particles (PM10)65. Several other studies showed no association between PM and lung function in adults with asthma. Though Lagorio et al. had observed a negative association between PM and lung function in subjects with COPD, there was no association between PM and lung function decrement among those with asthma59. Another study including subjects with COPD and asthma showed no association between indoor or outdoor air quality on lung function66. A study from Italy by Maestrelli et al. followed adult subjects with asthma periodically over 2 years and showed that measured personal exposure to PM10 during the 24 hours prior to assessment was associated with respiratory symptoms, but not FEV167.\n\nGiven the relatively small size of studies of air pollution on short-term changes in lung function in adults with COPD and asthma, they may not have been adequately powered to detect an association between pollutant exposure and lung function. Alternatively, it is possible that the adverse effects of PM exposure are linked to changes in smaller caliber airways that are not adequately captured by spirometric measures such as FEV1.\n\n\nEffect of PM reduction on lung function\n\nWhile the observational findings linking higher PM to worse lung function are intriguing, there is a need to provide additional levels of evidence, including assessment of temporality and observation as to whether lung function improves with reduction of PM exposure, to establish causality. Importantly, studies have shown that a decrease in pollution can improve lung function in both children and adults. For example, the Children’s Health Study in southern California followed three separate cohorts of children during periods of increasing PM regulation and therefore decreasing average PM concentrations. Investigators found higher FEV1 and FVC in the children exposed to the lowest concentrations of PM and, as air quality improved, the proportion of children with clinically low FEV1 subsequently declined68. Similarly, reductions in PM in an urban area of the Netherlands were associated with a 3% and 6% improvement in FEV1 and FVC, respectively, in both children and adults69. In the SAPALDIA study, a 10 μg/m3 decrease in PM10 concentration was associated with a 9% decrease in the rate of yearly decline of FEV1 in over 9000 randomly selected Swiss adults70. These studies highlight that even modest reductions in PM can translate to meaningful improvements in lung function.\n\nAs the EPA does not regulate the indoor environment, interventions to mitigate exposure to indoor PM are less well studied. A small number of research studies have shown improvements in indoor PM concentrations following behavioral modifications and use of indoor air cleaners71–73. Most of these studies did not assess the impact of these modifications on lung function, and the results are inconsistent in the ones that do. In one study of 48 nonsmoking adults, air cleaners placed in the living room and bedroom decreased PM2.5 concentrations but did not improve lung function in these participants, although conclusions may be limited by the small sample size74. In a randomized study of active versus sham air cleaners in 35 healthy college students in China, 48 hours of air purification significantly reduced indoor PM2.5 concentration by 57%. This decrease was associated with a significant reduction in circulating inflammatory markers, along with a non-significant trend towards slight improvement in lung function following air purification75. Air cleaners that were run for 1 week in 20 homes on the First Nations reserve in Canada significantly decreased indoor PM2.5 concentrations, a reduction that was associated with a 217 mL increase in FEV1 in 37 healthy individuals living in the homes76. These studies highlight the potential for indoor air modification to reduce total PM exposure; whether the improvement in indoor air quality can lead to improvements in lung function is yet to be determined.\n\n\nPotential mechanisms of susceptibility to PM\n\nThe precise mechanism as to how PM may influence health and lung function is unknown. Studies have suggested that PM may mediate adverse health effects via the generation of reactive oxygen species77–79, activation of cell signaling pathways, and alterations of respiratory tract barrier function and antioxidant defenses, all of which may lead to airway inflammation and changes in pulmonary function80. Additionally, cellular changes resulting from PM exposure may cause epigenetic modifications, leading to alterations in gene expression81. For example, results from the ENVIRONAGE birth cohort showed that PM2.5 exposure during gestation was associated with placental mitochondrial DNA methylation in 381 mother-newborn pairs82. These studies may offer insight into how PM exposure in utero may impact lung function later in life. Furthermore, activation of many cellular signaling pathways has been attributed to specific chemical and metal constituents of PM that have been isolated in vitro. This suggests that PM of various sources may lead to diverse responses, making compositional analysis of PM by region an important consideration for future research81,83.\n\nPatient factors, including demographic and genetic factors, may modify the impact of PM exposures on lung function. Our review reinforces the conclusion that PM exposure can have important impacts on lung function in those with and without existing lung disease; however, whether individuals with pre-existing respiratory disease are more susceptible to the adverse effects of PM exposure is unclear. Few studies examine the variability in lung function response to PM exposure specifically by respiratory disease status. Toxicological studies suggest that the presence of allergic airway conditions may increase susceptibility to PM exposure; however, epidemiologic studies have been inconsistent in clarifying this relationship84. For example, in Koenig et al.’s study of Seattle schoolchildren, PM exposure impacted lung function in only those children with asthma, not healthy children38. Conversely, in a study of Mexico City schoolchildren, decrements in FVC following higher exposure to PM2.5 were seen in both the 158 asthmatic children and the 50 children without asthma85. Similarly, individuals with COPD may have impairment in mucociliary clearance, which may lead to an increase in dose of fine particles86 and resultant greater risk of PM-related respiratory effects, although epidemiological studies are needed to confirm this hypothesis84. Additionally, several studies have suggested that the impact of PM on lung function may vary by gender, and early life may be a critical time window when PM may adversely affect lung function. A few studies have also investigated whether genetic polymorphisms may modify the effects of PM on lung function. For example, results from Breton et al. suggest that genetic variations in the glutathione synthesis pathway may modify the impact of PM exposure on lung function in children87 and several investigations from the SAPALDIA cohort suggest that genetic variations potentially mediate the effect of PM on lung function decline88,89. Identifying risk factors of those who will be more susceptible to the health effects of PM exposure is a research priority.\n\n\nConclusion\n\nIn summary, the existing research suggests that PM exposure can influence lung development and have an important impact on lung function in both children and adults, and in those with and without existing lung disease. Although not without inconsistencies, this research adds to the wider body of literature that supports an association between PM exposure and worse respiratory symptoms. To address the knowledge gaps that persist, large cohort studies with frequent comprehensive participant evaluations and individual-level PM exposure assessments are needed. In particular, studies on indoor air pollution have tended to be small, making definitive conclusions more challenging. In addition, size and composition of PM may have differing health effects; however, these differential effects have not yet been clearly elucidated. Lastly, there are several factors that may mediate the effects of PM exposure on lung health, such as timing of exposure, chronic lung diseases (including but not limited to obstructive airway diseases), genetics, and even other exposures such as medication use. Importantly, the studies to date suggest that relatively low concentrations of PM can negatively impact lung function, suggesting that meaningful health outcomes occur following exposure to relatively modest pollutant concentrations, many of which fall below the current EPA limits. Furthermore, while the observed decrements in lung function are relatively small, they have the potential to be clinically meaningful, especially in children, as those with lower lung function have been shown to have an increased risk of developing asthma90.\n\nMoreover, the improvement in lung function seen following PM reduction implies that the majority of the population will benefit from continued regulation of PM levels. In addition, the importance of short-term exposures is reflected in EPA standards that regulate not only average annual PM concentration but also 24-hour concentrations. To reflect the growing body of evidence suggesting the adverse impact of short-term PM exposures, these standards were strengthened in 20066. Policies and methods to decrease PM exposure are crucial to minimize the health impact of continued PM exposure on lung health. Due to the regulatory programs delineated as part of the original 1970 Clean Air Act and the 1990 amendments, there has been a 34% decrease in the annual national average of PM91. It is hoped that continued regulation of PM will help alleviate the impact that exposure has on the developing and adult lung.", "appendix": "Competing interests\n\n\n\nThe authors have no competing interests to disclose.\n\n\nGrant information\n\nLMP is supported by NIH grant K23ES025781, NNH is supported by NIH grants R01ES023500, R01ES018845, R01ES022607, P50MD010431, P01ES018176/ RD83451001, and P50ES015903.\n\n\nAcknowledgements\n\nThe authors would like to thank Carrie Price of the Welch Medical Library for her assistance in searching the published literature on this topic.\n\n\nReferences\n\nSilva RA, West JJ, Zhang Y, et al.: Global premature mortality due to anthropogenic outdoor air pollution and the contribution of past climate change. Environ Res Lett. 2013; 8(3): 34005. Publisher Full Text\n\nLelieveld J, Evans JS, Fnais M, et al.: The contribution of outdoor air pollution sources to premature mortality on a global scale. Nature. 2015; 525(7569): 367–71. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nImboden M, Schwartz J, Schindler C, et al.: Decreased PM10 exposure attenuates age-related lung function decline: genetic variants in p53, p21, and CCND1 modify this effect. Environ Health Perspect. 2009; 117(9): 1420–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIslam T, Gauderman WJ, Berhane K, et al.: Relationship between air pollution, lung function and asthma in adolescents. Thorax. 2007; 62(11): 957–63. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOffice of Air and Radation, U.S. Enivronmnetal Protection Agency, Washington DC: The Benefits and Costs of the Clean Air Act from 1990 to 2020: Summary Report 2011. Reference Source" }	[ { "id": "12572", "date": "22 Feb 2016", "name": "Alfésio Luís Ferreira Braga", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12571", "date": "22 Feb 2016", "name": "Stephan van Eeden", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12581", "date": "22 Feb 2016", "name": "Xinbiao 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", "responses": [] } ]	1	https://f1000research.com/articles/5-201
https://f1000research.com/articles/5-195/v1	19 Feb 16	{ "type": "Opinion Article", "title": "Tripartite genome of all species", "authors": [ "MengPing Long", "TaoBo Hu" ], "abstract": "Neutral theory has dominated the molecular evolution field for more than half a century, but it has been severely challenged by the recently emerged Maximum Genetic Diversity (MGD) theory. However, based on our recent work of tripartite human genome architecture, we found that MGD theory may have overlooked the regulatory but variable genomic regions that increase with species complexity. Here we propose a new molecular evolution theory named Increasing Functional Variation (IFV) hypothesis. According to the IFV hypothesis, the genome of all species is divided into three regions that are ‘functional and invariable’, ‘functional and variable’ and ‘non-functional and variable’. While the ‘non-functional and variable’ region decreases as species become more complex, the other two regions increase.", "keywords": [ "Increasing Functional Variation hypothesis", "Maximum Genetic Diversity theory", "Neutral theory", "Evolution", "Genome architecture", "Gene regulation" ], "content": "Introduction\n\nThe structure and function of the genome have been a major question that all researchers want to solve. The current popular view of the genomic structure is represented by the neutral theory. The neutral theory states that the majority of the genome is variable and neutral1. The variable property of these genomic regions would not change as the complexity of species increases (Figure 1).\n\nWhile the neutral theory and MGD theory analyze genome structure as bipartite, the IFV hypothesis adds an additional region which is the variable and functional gene regulatory region. As species complexity increases, the variable region of the genome would stay as variable according to neutral theory. While in MGD theory, as species complexity increases there would be less variable region. Unlike MGD theory, IFV hypothesis states that the functional variable region which contains gene regulatory elements would also increase with species complexity.\n\nWhile in recent years, another theory called Maximum Genetic Diversity (MGD) provided unprecedented insights into the genome structure2–5. The MGD theory originated from blasting some conserved proteins such as cytochrome C and hemoglobin of different species. By computing the changeable sites of each species3, Huang found that more complex species have less changeable sites in certain regions of the genome. Thus, MGD theory states that as the complexity of species increases, the genome would have more invariable regions and less variable regions (Figure 1).\n\n\nIFV hypothesis\n\nHere we proposed the Increasing Functional Variation (IFV) hypothesis inspired by both the MGD theory2 and our recent work on human genome architecture6. Recently, based on co-localization of various genomic features we divided the human genome into three parts, referred to as gene enriched (Genic) zones, gene regulatory elements enriched (Proximal) zones and non-functional features enriched (Distal) zones6. We regard the Genic zones as mainly functional and invariable, and the Distal zones as mainly non-functional and variable. The Proximal zones that compose 31% of human genome contain the majority of gene regulatory elements including transcriptional factor binding sites (TFBSs) and are at the same time enriched with conserved indels. These features make Proximal zones functional and variable. It has been proven that as the complexity of species increase, there would be more gene regulatory region in the genome. Based on these two points, we propose that as the complexity of species increases, this variable part of the genome which contains functional regulatory elements would also increase. We call it the Increasing Functional Variation (IFV) hypothesis. Besides the variable gene regulatory region, the other part of the genome can be divided into two parts, the functional and invariable region and the non-functional and variable region. The alteration of these two parts with species complexity can be explained by MGD theory (Figure 1). What the MGD theory lacks and IVF hypothesis complements is the existence of the variable and functional gene regulatory region in the genome. And according to the IFV hypothesis, as species complexity increases, the variable part of the genome would not simply decrease as stated by MGD theory. The differences between IVF hypothesis and MGD theory have been illustrated in Table 1.\n\n\nConclusions\n\nMGD theory has refuted the idea stated by the neutral theory that the majority of the genome is neutral and variable among all species. Instead, it proved that the variable region of the genome would decrease as species become more complex.\n\nHowever, MGD theory has its own limitation as pointed out by Ho shortly after the publication of MGD. As Ho has mentioned in her book7, more complex species have more sequence diversity, which is needed for precise regulation of local somatic expression. Ho also stated that although MGD theory solved the paradoxes in molecular evolution, the diversity of complex species at somatic level can’t be explained by it. Our recent study6 on human genome architecture discovered not only variable but also functional regions of the human genome. In an attempt to provide a more comprehensive view of genome structure and molecular evolution, we developed the IFV hypothesis based on our discovery of the variable property of the gene regulatory region.\n\nWhy would we develop this tripartite model of genome architecture across all species? As the ancient Chinese philosopher Lao Tzu stated in Tao Te Ching thousands of years ago that “three engenders the myriad things”, which means “three” is the root of all things. If the truth of the universe is universal, we believe that the consistency between our tripartite genome architecture of all species and Lao Tzu’s philosophical thinking is not a coincidence.", "appendix": "Author contributions\n\n\n\nTBH & MPL jointly conducted this work and wrote the manuscript.\n\n\nCompeting interests\n\n\n\nThe authors declare there is no conflict of interest.\n\n\nGrant information\n\nMPL & TBH are recipients of MD-PhD scholarship from Hong Kong University of Science and Technology. This work was supported by the grant to TBH from Central South University, China (2282013bks118).\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nNei M, Suzuki Y, Nozawa M: The neutral theory of molecular evolution in the genomic era. Annu Rev Genomics Hum Genet. 2010; 11: 265–89. PubMed Abstract \| Publisher Full Text\n\nHu T, Long M, Yuan D, et al.: The genetic equidistance result: misreading by the molecular clock and neutral theory and reinterpretation nearly half of a century later. Sci China Life Sci. 2013; 56(3): 254–61. PubMed Abstract \| Publisher Full Text\n\nHuang S: The Overlap Feature of the Genetic Equidistance Result—A Fundamental Biological Phenomenon Overlooked for Nearly Half of a Century. Biological Theory. 2010; 5(1): 40–52. Reference Source\n\nHuang S: Inverse relationship between genetic diversity and epigenetic complexity. Preprint available at Nature Proceedings. 2009. Publisher Full Text\n\nHuang S: The Genetic Equidistance Result of Molecular Evolution is Independent of Mutation Rates. J Comput Sci Syst Biol. 2008; 1: 92–102. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNg SK, Hu T, Long X, et al.: Feature co-localization landscape of the human genome. Sci Rep. 2016; 6: 20650. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHo MW: Development and Evolution Revisited. In: Hood KE, Halpern CT, Greenberg G, Lerner RM, editors. Handbook of Developmental Science, Behavior, and Genetics. Blackwell Publishing Ltd; 2010. Publisher Full Text" }	[ { "id": "12943", "date": "29 Mar 2016", "name": "Shaillay Dogra", "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 would be nicer for the readers if there was more explanation and discussion in the text on the various different theories, what they imply, their pros and cons etc. Currently, it requires some background reading on these topics for the not so well-informed reader.A detailed discussion of the basis of the IVF hypothesis, the basis of the proposals, its criticism etc. is suggested for a greater understanding of the author's ideas.", "responses": [] }, { "id": "13461", "date": "03 May 2016", "name": "Rahul Banerjee", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 Long and Hu is of considerable interest in the field of evolution of genomes. However the authors could consider the following points to improve the quality of the manuscript :Is there any numerical measure of species complexity ? On page 2, column 2, line 6'Huang found that more complex species have less changeable sites in certain regions of the genomes.'It would be nice if some more details could be included with regard to these sites. The authors should be more elaborate as to why they considered the Genic zones to be mainly functional and invariable and the Distal zones as mainly non-functional and variable, and the Proximal zones functional and variable. The note about Lao Tzu was also quite attractive and added some philosophical charm to the scientific note.", "responses": [] }, { "id": "14116", "date": "16 Jun 2016", "name": "Alfredo Pulvirenti", "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, in its current version, presents several major limitations.\n\nThe authors should provide more details about the theories.\nFurthermore, the rationale behind their IFV hypothesis should be given in a deeper way. Examples supporting the conclusions are strongly needed. The authors should also highlight the limitations and the opportunities of this hypothesis.", "responses": [] } ]	1	https://f1000research.com/articles/5-195
https://f1000research.com/articles/5-193/v1	18 Feb 16	{ "type": "Research Note", "title": "Expression of the retina-specific flippase, ABCA4, in epidermal keratinocytes", "authors": [ "Luke A. Wiley", "Emily E. Kaalberg", "Jessica A. Penticoff", "Robert F. Mullins", "Edwin M. Stone", "Budd A. Tucker", "Luke A. Wiley", "Emily E. Kaalberg", "Jessica A. Penticoff", "Robert F. Mullins", "Edwin M. Stone" ], "abstract": "ATP-binding cassette, sub-family A, member 4 (ABCA4) is a photoreceptor transmembrane protein that is responsible for flipping N-retinylidene-phosphatidylethanolamine, a key intermediate in the visual cycle, from the lumen to the cytoplasmic leaflet of photoreceptor outer segment disks. Mutations in ABCA4 cause a build-up of toxic retinoids resulting in a variety of retinal degenerative phenotypes, including Stargardt disease, cone-rod dystrophy and retinitis pigmentosa. Since many of the ABCA4 variants are rare and non-exomic, their pathogenicity is often difficult to demonstrate statistically. Given that the neural retina is inaccessible to molecular analysis in living patients, we use patient-specific induced pluripotent stem cell (iPSC)-derived retinal neurons to identify and model disease-causing mutations. Here we demonstrate that a truncated version of the retinal-specific transmembrane enzyme ABCA4 is expressed in epidermal keratinocytes and is required for cellular proliferation and viability at late passage. This finding is of great importance for labs that wish to investigate the pathophysiology of novel ABCA4-variants, without having to incur the added expense and scientific expertise associated with iPSC generation, culture and differentiation. Likewise, this finding is also important for those intending to generate iPSCs from patient specific keratinocytes, which can prove difficult when ABCA4 mutations are present.", "keywords": [ "ABCA4", "induced pluripotent stem cells", "keratinocytes" ], "content": "Narrative\n\nWe have demonstrated that a truncated isoform of the retina-specific protein, ABCA4 is expressed in human epidermal keratinocytes. This isoform is required for cell viability at late passage and as such can be used to interrogate the pathophysiology of novel ABCA4 mutations.\n\n\nIntroduction\n\nATP-binding cassette, sub-family A, member 4 (ABCA4, previously named ABCR for its retina-specific expression) is a transmembrane protein that is primarily localized to retinal photoreceptors1 where it is responsible for flipping N-retinyldene-phosphatidylethanolamine, a key intermediate in the visual cycle, from the lumen to the cytoplasmic leaflet of photoreceptor outer segment disks1,2. Mutations in ABCA4 cause a build-up of toxic retinoids resulting in a variety of retinal degenerative phenotypes, including Stargardt disease, cone-rod dystrophy and retinitis pigmentosa2. To date over 400 different mutations in ABCA4 have been reported3. Since many of these variants are rare and non-exomic, their pathogenicity is often difficult to prove4. As the primary focus of our group is to develop gene and autologous cell replacement-based treatments for rare inherited retinal degenerative diseases, unambiguous identification of disease-causing mutations is essential.\n\nThe neural retina is inaccessible to molecular analysis in living patients. As such, to determine if newly identified mutations in retina-specific genes are disease causing, we use patient-specific induced pluripotent stem cell (iPSCs)-derived retinal neurons5. Typically, iPSCs are generated in our lab using patient-specific keratinocytes isolated from the epidermis of 3 mm punch biopsies. Briefly, the epidermis is separated from the underlying dermis via dispase incubation, and keratinocytes are liberated via trituration. Keratinocytes are subsequently maintained on collagen-coated culture plates in Epilife medium (Gibco; Life Technologies) supplemented with 1% human Keratinocyte Growth Supplement (Gibco). While generating iPSCs from patients suspected of having ABCA4-associated retinal disease, we noticed that primary keratinocyte proliferation was often slow and reprograming efficiencies were low compared to age-matched controls, suggesting that ABCA4 mutations affect keratinocyte physiology directly. In this study a series of western blot, immunocytochemistry, and cytometry experiments were performed to demonstrate that ABCA4 is expressed in human keratinocytes and that ABCA4 mutations alter cellular viability.\n\n\nResults\n\nTo determine if the ABCA4 protein was expressed in keratinocytes, a Western blot comparing lysates isolated from normal human, bovine and murine retina, murine retina with Abca4 deletions, and human keratinocytes was performed. Keratinocytes from three different control individuals of three different ages expressed a truncated protein that corresponded in size to an alternatively spliced 70 kDa ABCA4 isoform (detected previously via rt-PCR4; (Figure 1A)). Immunocytochemical labeling using an antibody targeted against ABCA4 demonstrated a perinuclear pattern of labeling (Figure 1B–C). To determine if mutations in ABCA4 affect cellular proliferation and/or senescence, keratinocytes from three control, three retinal disease patients without ABCA4 mutations, and three patients with molecularly confirmed ABCA4-associated retinal disease were analyzed using propidium iodide and a Tali image based cytometer (Figure 1D). For this analysis, keratinocytes received fresh medium every other day and were passaged routinely once a week. Cell viability readings were assessed at each cell passage. A significant decrease in cell viability was detected between passages 6–9 in cells isolated from patients with ABCA4-associated disease as compared to normal and non-ABCA4 disease controls (Figure 1D). Finally, immunocytochemical staining of normal human skin shows robust ABCA4 expression throughout the keratinizing layer (Figure 1E).\n\nA) Western blot of human (Hmn Ret), bovine (Bov Ret) and murine (Ms Ret) whole retinal lysate, retinal lysate from two mouse strains lacking Abca4 (Abca4 KO and Japanese Fancy (JF1)) and keratinocytes from three individuals 32, 54 and 68 years of age (KTC 32, 54, and 68, respectively). Robust ABCA4 expression is observed at 250 kDa in human, cow and mouse retina, but is completely absent in each Abca4 knockout retina. Keratinocytes from each control individual express a 70 kDa isoform of ABCA4. B–C) Immunocytochemical labeling of control KTC 32 (B) and control KTC 54 (C) patient keratinocytes with an anti-ABCA4 antibody (red) and a filamentous actin stain (Phalloidin; green). A perinuclear pattern of ABCA4 labeling was detected. D) Keratinocyte viability was assessed at passages 6–9 by staining cells with propidium iodide. Keratinocytes assessed from three different individuals with molecularly confirmed mutations in ABCA4 (“ABCA4”) are much less viable compared to control keratinocytes (“control”) and cells from three different patients with non-ABCA4-associated retinal disease (“Non-ABCA4”). E) Immunocytochemical staining of control human skin with the same anti-ABCA4 antibody used in Figure 1A–C above showing ABCA4 labeling (green) throughout the keratinizing layer of the epidermis. Scale bars = 100 μm (B–C) and 40 μm (E).\n\n\nConclusions\n\nTaken together, these data demonstrate that a truncated version of the retinal-specific transmembrane enzyme ABCA4 is expressed in epidermal keratinocytes and is required for cell viability at late passage. Although future studies will be needed to elucidate the exact function of ABCA4 in the skin, this finding may be useful for individuals attempting to determine the pathogenicity of novel mutations in the ABCA4 gene.\n\n\nMaterials and methods\n\nAll patients provided written, informed consent for this study, including skin biospies and use of keratinocytes to generate induced pluripotent stem cells, which was approved by the Institutional Review Board of the University of Iowa (project approval #199904167) and adhered to the tenets set forth in the Declaration of Helsinki.\n\nWestern blotting was performed as previously described5–7. Briefly, 40 μg of protein lysate from unaffected human donor retina (Iowa Lions Eye Bank, Coralville, IA), bovine retina (Bud’s Custom Meats Inc., Riverside, IA), control mouse retina (C57Bl/6J; The Jackson Laboratory, Bar Harbor, ME; Cat. No. 00064), Abca4-/- (KO; a kind gift from Dr. Gabriel Travis, University of California Los Angeles), Japanese Fancy mouse retina (a kind gift from Dr. Toshihiko Shiroishi, National Institute of Genetics, Mishima, Japan) and keratinocytes from three individuals ages 32, 54 and 68 were separated via SDS-PAGE on a 4–20% gradient gel, blotted and labeled with a mouse monoclonal anti-ABCA4 antibody at a 1:500 dilution (Santa Cruz Biotechnology, Dallas, TX; Cat. No. sc-65672). Bands were visualized using SuperSignal® West Pico Chemiluminescence Substrate (Thermo Fisher Scientific, Waltham, MA; Cat. No. 34080) and autoradiography. Images of the final blots were captured with an iPhone 6S (Apple, Cupertino, CA).\n\nKeratinocytes were fixed for 10 minutes in 4% paraformaldehyde in 1X phosphate buffered saline (PBS), rinsed in 1X PBS and blocked in immunocytochemical blocking buffer [1X phosphate buffered saline (Cat. No. 10010-023; Thermo Fisher Scientific, Waltham, MA, USA), 3% bovine serum albumin (Cat. No. A30075-100.0; Research Products International Corp., Mount Prospect, IL, USA), 5% normal goat serum (Note: another species can be substituted here to better suit the secondary antibody species; Cat. No. 5425; Cell Signaling, Danvers, MA, USA), 0.5% Triton X-100 (Cat. No. T8787; Sigma-Aldrich, St. Louis, MO, USA), and 0.2% NaN3 (sodium azide; Cat. No. 438456; Sigma-Aldrich, St. Louis, MO, USA)]. Cells were labeled with mouse monoclonal anti-ABCA4 antibody at a dilution of 1:500 (Santa Cruz Biotechnology, Dallas, TX; Cat. No. sc-65672) and stained with Alexa Fluor® 488 Phalloidin at a dilution of 1:1000 (Life Technologies/Thermo Fisher Scientific, Waltham, MA; Cat#: A12379) to label filamentous actin overnight at 4° Celsius. The following morning, cells were washed using wash buffer [1X phosphate buffered saline (Cat. No. 10010-023; Thermo Fisher Scientific, Waltham, MA, USA), 0.2% Tween® 20 (Cat. No. P2287; Sigma-Aldrich, St. Louis, MO, USA)]. ABCA4 was detected using the goat anti-mouse 555 fluorescently conjugated Alexa Fluor® secondary polyclonal antibody at a dilution of 1:1000 (Life Technologies/Thermo Fisher Scientific, Waltham, MA; Cat#: A-21422) for two hours at room temperature. Cell nuclei were coverslipped and counterstained using Poly(vinyl alcohol) (PVA)-based mounting medium containing 1,4-Diazabicyclo[2.2.2]octane (DABCO) [100 μg/ml PVA (Cat. No. 341584; Sigma-Aldrich, St. Louis, MO, USA), 25% v/v glycerol (Cat. No. G9012; Sigma-Aldrich, St. Louis, MO, USA), 0.1M Tris-HCl, pH 8-8.5, 25 μg/ml DABCO (Cat. No. 290734; Sigma-Aldrich, St. Louis, MO, USA) and 4’,6-Diamidino-2-phenylindole dihydrochloride (DAPI) (Cat. No. D9542; Sigma-Aldrich, St. Louis MO, USA; 1:10,000 dilution). Cells were imaged using a Leica DM 2500 SPE confocal microscope (Leica Microsystems, Wetzlar, Germany). Human skin was labeled with anti-ABCA4, secondary antibody and visualized in the same manner.\n\nTo determine if mutations in ABCA4 affect cellular proliferation and viability, keratinocytes from three control, three retinal disease patients without ABCA4 mutations, and three patients with molecularly confirmed ABCA4-associated retinal disease were analyzed using propidium iodide (Thermo Fisher Scientific, Waltham, MA; Cat. No. P3566) and a Tali image-based cytometer (Life Technologies, Waltham, MA). Keratinocytes were maintained on collagen-coated culture plates in Epilife medium (Gibco/Life Technologies, Waltham, MA; Cat. No. M-EPI-500-CA) supplemented with 1% Human Keratinocyte Growth Supplement (Gibco/Life Technologies, Waltham, MA; Cat. No. S-001-5). Keratinocytes received fresh medium every other day and were passaged routinely once a week. Cell viability readings were obtained using the Tali image-based cytometer via PI staining and 20 image analysis, i.e. at the time of passage a sample from each cell line was loaded into the Tali, 20 separate images were taken and the number of live vs. dead cells and in turn % viability was automatically generated. This data was collected from cell lines obtained from 7 control individuals, 5 patients with non-ABCA4-associated retinal disease (disease controls) and 13 patients with two molecularly confirmed disease causing ABCA4 mutations. To determine statistical significance, a one-way ANOVA with a Tukey’s post-hoc test was performed use Graphpad Prism software version 6.0b (Graphpad Software Inc., La Jolla, Ca). A p-value < 0.05 was considered significant.\n\n\nData availability\n\nF1000Research: Dataset 1. Raw data for ‘Expression of the retina-specific flippase, ABCA4, in epidermal keratinocytes’, 10.5256/f1000research.8089.d1140778", "appendix": "Author contributions\n\n\n\nLAW, RFM, EMS and BAT conceived the study. LAW, EEK and JAP performed the experiments. LAW, RFM, EMS and BAT wrote and approved the final manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nNIH Directors New Innovator Award 1-DP2-OD007483-01 (BAT); NEI EY016822 (EMS); NEI EY024605 (RFM); Research to Prevent Blindness; Stephen A. Wynn Foundation.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nWeng J, Mata NL, Azarian SM, et al.: Insights into the function of Rim protein in photoreceptors and etiology of Stargardt's disease from the phenotype in abcr knockout mice. Cell. 1999; 98(1): 13–23. PubMed Abstract \| Publisher Full Text\n\nSheffield VC, Stone EM: Genomics and the eye. N Engl J Med. 2011; 364(20): 1932–42. PubMed Abstract \| Publisher Full Text\n\nTsybovsky Y, Molday RS, Palczewski K: The ATP-binding cassette transporter ABCA4: structural and functional properties and role in retinal disease. Adv Exp Med Biol. 2010; 703: 105–25. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBraun TA, Mullins RF, Wagner AH, et al.: Non-exomic and synonymous variants in ABCA4 are an important cause of Stargardt disease. Hum Mol Genet. 2013; 22(25): 5136–45. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTucker BA, Mullins RF, Streb LM, et al.: Patient-specific iPSC-derived photoreceptor precursor cells as a means to investigate retinitis pigmentosa. eLife. 2013; 2: e00824. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTucker BA, Scheetz TE, Mullins RF, et al.: Exome sequencing and analysis of induced pluripotent stem cells identify the cilia-related gene male germ cell-associated kinase (MAK) as a cause of retinitis pigmentosa. Proc Natl Acad Sci U S A. 2011; 108(34): E569–76. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBurnight ER, Wiley LA, Drack AV, et al.: CEP290 gene transfer rescues Leber congenital amaurosis cellular phenotype. Gene Ther. 2014; 21(7): 662–72. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWiley L, Kaalberg E, Penticoff J, et al.: Dataset 1 in: Expression of the retina-specific flippase, ABCA4, in epidermal keratinocytes. F1000Research. 2016. Data Source" }	[ { "id": "12898", "date": "04 Apr 2016", "name": "Iain S. Haslam", "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 some novel data showing the expression of a truncated version of the ABCA4 protein in primary human keratinocytes. They determine that primary keratinocytes from patients with retinal disease-causing ABCA4 mutations have lower viability at later passage. The article is generally well written and the data is novel and interesting, however I would recommend addressing some points I raise below.Abstract.The final sentence a little vague. Without having read the paper this statement is not too clear. As the abstract should draw the reader into the article, a better summarising/concluding statement should end the abstract. Also, the authors state that ABCA4 is important for cellular proliferation. Their paper does not however provide any measure of proliferation so they should only state that viability is affected, in the absence of such data. Figure 1AWestern blot bands prominent but rather spread-out, particularly in the ms retina. What do the other bands present correspond to, are they non-specific? Other known splice variants? Do the authors know the antibody epitope and are they confident it cross-reacts appropriately with ABCA4 in different species? Figure 1DCells from how many patients were used for each measurement? The figure legend suggests 3, but the materials and methods states different numbers of patients/donors for each condition. This needs clarifying in the figure legend. Also, why no indication of statistical significance on the graphs? The authors claim to have performed one-way ANOVA but don’t show the result in the figure? Figure 1B/C What was the passage of the cells used to generate these images and are they matched? The image in Figure 1C appears to have many more large, senescent cells in which the staining pattern appears less perinuclear and more punctate, throughout the cytoplasm. Can the authors provide more details and comment on this? Figure 1E The DAPI staining appears absent in the basal layer in places, why is this? The description of the staining is somewhat inadequate. What about subcellular localisation? In the basal layer it almost looks membraneous in places, however as you move through the stratum spinosum and granulosum, the staining becomes strongly and uniformly cytoplasmic. This Figure would benefit from additional annotation, showing the dermal-epidermal junction and highlighting the different layers of the epidermis. This would help readers identify the changes in localisation that appear to occur as the keratinocytes differentiate. ConclusionsGiven the authors finding that ABCA4 is expressed in keratinocytes, plus my own previously published findings that it is also strongly expressed in human hair follicles, is it appropriate to still call ABCA4 “retina-specific”? I would say not. Can the authors expand on exactly how and why this study will be useful for determining the pathogenicity of novel ABCA4 mutations? Overall – I understand that iPSCs derived from epidermal keratinocytes are used as a retinal surrogate in lieu of being able to access retina’s from living patients. I now understand that keratinocytes express a truncated version of ABCA4, which has some unknown role in maintaining the viability of the isolated primary cells. What is not clear to me is how this can help with studying the pathogenicity of ABCA4 mutations? If only a truncated version of ABCA4 is present in keratinocytes, how does this form relate to the full-length protein? And can the mutations that are commonly found in patients with retinal disease be mapped to this truncated version? Overall I think this is really interesting phenomenological data, but for me the authors do not clearly describe how their findings could be useful for studying novel mutations in full-length ABCA4, as would be present in the retina? It would be really useful to have some sequencing data that might show differences in the truncated ABCA4 present in the primary keratinocytes from control vs. stargardt patients.", "responses": [ { "c_id": "1937", "date": "21 Apr 2016", "name": "Luke Wiley", "role": "Author Response", "response": "We thank both reviewers for their insightful comments and critique of this brief manuscript. We agree with the comments from each reviewer, particularly the additional experiments that each suggested, which would act to strengthen the paper. However, we felt this was an important observation to share in an open publishing forum, so as to reach as many people as possible. Again, while we agree with and appreciate each reviewer's critique, to address all of these comments would be too time consuming for a brief observational report such as this." } ] }, { "id": "13220", "date": "20 Apr 2016", "name": "Roxana A. Radu", "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 Wiley et al. presents an interesting and novel finding that keratinocytes express a truncated form of ABCA4 protein that may be responsible for cell viability. Keratinocytes from patients with disease-causing mutations in the ABCA4 protein appear to have low viability upon passaging in culture when compared to control cells (from patients with unknown ABCA4 mutations and unaffected individuals). The manuscript is well written and the data are clearly presented. However, there are some important key points that are not fully clear thereby making it difficult to accurately assess the biological relevance of the current work:Fig. 1 panel A: an internal control should be considered in order to estimate the relative abundance of the ABCA4 isoform relative to the retina samples. Although the authors do not comment on this aspect, there is a ~70kDA band in both bovine and human retina samples. Perhaps, lowering the total protein amount and including an internal standard (i.e. tubulin, actin or GAPDH) along with keratinocytes from patients with disease-causing ABCA4 mutations could help with interpreting and quantifying the data from this panel. Fig. 1 panel B and C: at gross evaluation, there are slight differences between the distribution of ABCA4/Phalloidin staining of the two analyzed normal keratinocytes. There are larger cells and more punctate ABCA4 distribution in KTC 54 (panel B) compared to KTC32 (panel C), possibly due to different passages or culturing conditions. It may help to add the ABCA4/Phalloidin staining profiles of keratinocytes collected from patients with disease-causing ABCA4 mutations. All cells should be harvested, cultured and passaged similarly. Moreover, how specific is this ABCA4 antibody? A negative control, pre-immune (secondary antibody only) must be included to determine the background staining level. Fig. 1 panel D: representative images of passages 6 and 9 should be provided for each group. The identification of disease-causing ABCA4 mutations found in the studied keratinocytes should be reported.\n\nFig. 1 panel E: ABCA4 immunostaining should be done in the skin of the patients with molecularly confirmed ABCA4 mutations also not just in the control skin of a normal individual. General comment: It would be important to know if selected keratinocytes from patients with disease-causing ABCA4 mutations express a misfolded ABCA4 protein or if they do not express the ABCA4 protein at all. Providing sequencing data regarding the ABCA4 variants from keratinocytes isolated from Stargardt patients would be valuable.The discussion should be expanded to explain how the identification of this truncated form of ABCA4 is relevant to studies of the full-length ABCA4 in the iPS-derived photoreceptor cells or perhaps in other cell types. It would also be interesting to know if it is even possible to reprogram and differentiate these genetically mutated keratinocytes in order to obtain a specific cell-type that may be useful for future analyses. The identification of the ABCA4 protein in keratinocytes, most likely an alternatively spliced isoform, is novel and suggests a physiologic role of ABCA4 in other cell types besides the photoreceptors.", "responses": [ { "c_id": "1936", "date": "21 Apr 2016", "name": "Luke Wiley", "role": "Author Response", "response": "We thank both reviewers for their insightful comments and critique of this brief manuscript. We agree with the comments from each reviewer, particularly the additional experiments that each suggested, which would act to strengthen the paper. However, we felt this was an important observation to share in an open publishing forum, so as to reach as many people as possible. Again, while we agree with and appreciate each reviewer's critique, to address all of these comments would be too time consuming for a brief observational report such as this." } ] } ]	1	https://f1000research.com/articles/5-193
https://f1000research.com/articles/5-191/v1	18 Feb 16	{ "type": "Review", "title": "Signaling in T cells – is anything the m(a)TOR with the picture(s)?", "authors": [ "Mark Boothby" ], "abstract": "The excitement surrounding checkpoint inhibitors in the treatment of patients with cancer exemplifies a triumph of the long-term value of investing in basic science and fundamental questions of T-cell signaling. The pharmaceutical future actively embraces ways of making more patients’ cancers responsive to these inhibitors. Such a process will be aided by elucidation of signaling and regulation. With thousands of articles spread across almost 30 years, this commentary can touch only on portions of the canonical picture of T-cell signaling and provide a few parables from work on mammalian (or mechanistic) target of rapamycin (mTOR) pathways as they link to early and later phases of lymphocyte activation. The piece will turn a critical eye to some issues with models about these pathways in T cells. Many of the best insights lie in the future despite all that is uncovered already, but a contention is that further therapeutic successes will be fostered by dealing with disparities among findings and attention to the temporal, spatial, and stochastic aspects of T-cell responses. Finally, thoughts on some (though not all) items urgently needed for future progress will be mooted.", "keywords": [ "T-cell", "T-cell signalling", "mTOR" ], "content": "A recap of “T-cell activation” and the checkpoint inhibitor breakthrough\n\nCheckpoint inhibitors represent a breakthrough treatment class for patients with cancer1–3 and derive from investing in basic science and fundamental questions of T-cell signaling. What are the roots of this revolution and how does it relate to signaling inside T cells? On conceptual grounds, Bretscher and Cohn hypothesized that T-cell activation should require two signals4. Myriad studies established the principle that T cells would be activated through an antigen receptor and that this T-cell antigen receptor (TCR) was stimulated by encounter with a cell whose surface bears a specialized protein encoded in the major histocompatibility complex (MHC) combined with a suitable peptide5,6. TCR interactions with an appropriate MHC-peptide complex, however, could yield a state of unresponsiveness when an arcane chemical fixative was used to analyze antigen presentation7. These findings implied that TCR stimulation was not enough; a co-stimulatory molecule was required, consistent with the model by Bretscher and Cohn. A race to elucidate the mechanism culminated in identification of the CD28 co-stimulatory receptor as the protein stimulated by a fixation-sensitive ligand on the antigen-presenting cell (APC) displaying a stimulatory MHC-peptide complex8. Of note, few if any of these investigations were driven by “cancer research”.\n\nThis galaxy of investigation in turn led to families of co-stimulatory (4-1BB, ICOS, etc.) and co-inhibitory receptors (among which are CTLA-4 and PD-1, each a target of one class of pharmaceutical to enhance immune activity hitting tumors)9–11. In contrast to CD28, most of these crucial collaborators in TCR signal-interpretation are induced after T-cell activation. In parallel, the discovery of these regulators of T-cell activation, proliferation, and function prompted identification of their ligands, typically proteins expressed on the surfaces of APCs. These proteins that engage the co-stimulatory and -inhibitory receptors also follow a theme dividing constitutive (for instance, on so-called “professional APCs”) and inducible expression. Inflammation, including local cytokines but perhaps also intracellular sensors of cell stress (such as inflammasomes, Nod-like receptors, or cGAS), can promote induction of the ligands (e.g. PD-L1 or PD-L2) for the co-regulators (e.g. PD-1).\n\nHowever, much of the emergence of effector functions for a naïve T cell takes place days after it first starts being activated, and intra-vital imaging reveals striking journeys traveled by the motile T lymphocyte and its daughters after an activating encounter12–15. At present, sufficient breadth and depth of information about these variable and less deterministic aspects of T-cell population development are sorely lacking, in part because of limitations in the present state of technology for such analyses. Accordingly, a missing link and frontier for investigation must be the time element and dynamism of influences on the T cell even after its initial encounter with an APC bearing stimulatory peptide-MHC complexes and cell-scale resolution of the variegated nutritional environments in which T cells operate.\n\n\nDrilling in the message(s): TCR-activated signal transduction\n\nThe simple canonical outline of early signal transduction activated by TCR engagement and CD28 co-stimulation has been beautifully reviewed, most recently by Malissen16,17, who highlights points of uncertainty. In the general model, Src-family kinases initiate protein tyrosine phosphorylation at characteristic tyrosines in ITAMs (immunoreceptor tyrosine activation motifs) on the cytoplasmic tails of chains essential to the overall TCR complex. Phospho-ITAMs recruit adapter proteins crucial for signal propagation and diversification. One of these recruits is another protein tyrosine kinase, ZAP70, which phosphorylates yet further targets in the cascade. Of note, genetic and pharmacological analyses reveal ZAP70 as a prototype of a relay essential for one type of outcome from TCR engagement yet not another18–20. One adapter notable among the relays downstream from ZAP70, LAT, can interact with several distinct classes of further adapter and transducer proteins. This arrangement affords the capacity to diversify the nature of signal21–24. Elegant genetic experiments provide evidence of the essential nature of most of the proteins and even of distinct functions for specific tyrosines within their sequences21. As detailed in later sections, activity of multi-protein complexes containing the target of rapamycin (mTOR) serine-threonine kinase is induced downstream from these earliest and most rapid TCR-triggered signals.\n\nSuch monomorphic descriptions skip over vital parts of the body of data that probably are as crucial for the biology and the adaptive value of the system as its basic outline. First, let us consider the naïve T cell. Here, particular cases of individual TCR and their interactions with activating ligands indicated that quantitative and qualitative differences (the densities of peptide-MHC complexes and the exact sequence of the peptide) led not only to quantitative and qualitative differences in later-phase signaling (for instance, that of ERK MAP kinases) but also to altered effector fate and function25–29. A functional reinforcement of these fundamental findings emerged in elegant experiments seeking to sketch out the constraints on probabilities of an individual TCR yielding particular effector subsets in vivo by use of single-progenitor transfers30,31. In short, how a TCR signals varies according to the TCR. A second layer is that, even within the single-cell transfer results, the variance in distributions for a given TCR was substantial and probably reflects stochastic events that may even have a largely random origin.\n\nSo, an item for the future is to determine how much adaptive value in immunity builds on stochastic variation in the signaling from an individual TCR and the immune analogue of Heisenberg’s uncertainty principle. As a first step on this path, methods of single-cell analysis and modeling32–35 will be an exciting frontier. Older evidence showing that the diverse populations of memory T cells arising from a given TCR signal differently from their naïve progenitor population presents a third issue. In line with the points raised earlier in this paragraph, this picture is complicated by the fact that different results were obtained depending on the TCR. Finally, another exciting point is that the functional impact of a signal or its subcellular localization can depend on whether the TCR is on a suppressive, tumor-promoting regulatory T (Treg) cell or some other form of CD4 T cell36.\n\n\nPI3K-mTOR signaling and the T cell\n\nThe lipid kinase phosphatidylinositol 3-kinase (PI3K) is another signal transduction mechanism initiated by the TCR or the combination of TCR and co-stimulatory receptor engagement. Notably, co-stimulatory receptors (e.g. CD28 and, even more strongly, ICOS) enhance generation of the lipid product of PI3K (i.e. phosphatidylinositol 3,4,5 triphosphate, or PIP3)37–42. This in turn enhances activity of serine-threonine kinases, including PIP3-dependent kinase-1 (PDK1) and its targets AKT, protein kinases C (PKC), and SGK1, as well as mTOR41–43 (Figure 1). Conversely, there is evidence that engagement of at least some of the co-inhibitory receptors decreases PI3K and mTOR activity44 but also that they stimulate these pathways45. Ultimately, these signals change gene expression profiles; alterations in DNA-binding transcription factors or other layers in the machinery for transcriptional regulation are probably a major part of the mechanism for such changes. Members of a branch of the Forkhead box transcription factor family (i.e. FoxO1 and FoxO3) are notable targets of these pathways. In particular, FoxO nuclear export and cytosolic retention are prompted by phosphorylation. The kinases for this negative regulation of FoxO are AKT – as regulated by phosphorylation by both PDK1 and mTOR in one of its two functional complexes – and SGK141,42,46. SGK1, like AKT, is activated by mTORC2. Though discussed less here because of constraints on length and focus, pertinent inputs from the stimulation of cytokine receptors by the complex mixtures of their ligands in the micro-environments of T cells also impact the behavior (survival, proliferation, migration, and differentiation) of lymphocytes. In aggregate, the sentences and paragraphs formed from the rich lexicon of cytokines are sometimes distilled to consideration of “signal 3” to integrate with the graded signals of TCR (signal 1) and co-stimulation/co-inhibition (signal 2).\n\nDetails are outlined in the text.\n\nAll together, this framework provides insights into – or “explains” – co-regulation, the various functional states of T cells, and the amount of functional activity these functional classes may retain (for instance, in the setting of anti-tumor responses of T cells). A central element of the story involves the capacity of CD4+ T cells to form various helper subsets but also both thymus-derived and peripherally generated suppressive Treg cells. Growth of a range of cancers is promoted (Treg) or restrained (cytotoxic CD8 T cells and some of the effector subsets) by subtypes of T cell47–50. Experiments pushing signaling to or beyond extremes of the dynamic range in physiology provided the result that persistently activated AKT blocked Treg fate51. These findings provide the crystal nucleus for a model in which modulation of the levels of AKT and mTOR provides a means to regulate a balance between taking on the Treg “fate” and that of T helper effectors such as the Th17 cell. Such a model harmonized with evidence derived from elimination of the enzyme mTOR from T lineage cells or inactivation of mTOR complex 252,53; in the extreme, this created a remarkable imbalance in which the effector fate after activation of naïve CD4+ T cells was almost completely diverted into that of cells expressing the “master regulator of Treg formation”, FoxP352.\n\nIn broader settings such as infection or cancer biology, of course, accounting for the involvement of and impact on thymus-derived Treg cells is a vital issue. Clearly, dominant suppression remains essential even after initial establishment of immune repertoires, since elimination of Treg from the mature mouse unleashed auto-inflammation54. Short of killing off FoxP3-expressing cells54, fate-marking experiments suggest that suppressive function is retained if FoxP3 is eliminated after establishment of the Treg state, although there is some divergence of findings and controversy in this area55,56. In a likely link to AKT, however, gene deletion analyses of the transcription factors FoxO1 and FoxO3a provide evidence that these transcription factors each regulate the formation of thymus-derived Treg cells but also that complete loss of FoxO1 undermined the functional capacity of FoxP3+ suppressors57–59. At one level, then, the collective findings yield a beautiful picture. Interference with PI3K-activating co-stimulation must decrease AKT-driven FoxO phosphorylation, thereby promoting its nuclear localization and Treg function.\n\nMany further findings add paint to this model while also raising questions as to “who’s in charge of mTOR activity?” Most broadly, work of the past several years has increasingly highlighted that the capacity to supply amino acids to a juxta-lysosomal locale also appears crucial for activity of mTORC1 and pathways downstream from it60–63. Work with mouse systems provides evidence that G-protein-coupled receptors for complement fragments C3a and C5a are essential for Treg function and shows that most of the CD4 T cells’ mTOR activity is lost when both C3aR and C5aR are absent64,65. In parallel, a body of work with human CD4 T cells indicates that an accessory protein in the complement system, CD46, is particularly crucial for enhancing leucine uptake rates and mTORC1 activity a day after T-cell stimulation or co-stimulation by antibody cross-linking66. Though not tied definitively to FoxP3, the body of studies on complement and CD46 suggest that the suppressive activity yielded after activation is influenced through this pathway67. This work with human cells starts to get at one of the great gaps in the canon (i.e. the time element and an assessment of how mechanisms evolve within an individual T cell and its progeny as it moves through time and different locations). Thus, much of the evidence on signaling in T-cell activation focuses on time points that are quite early in relation to the time at which effector phenotype or an enhanced probability of memory fate arises. Work with mouse and human Treg cells emphasizes a vital role for the satiety-regulating hormone leptin in T-cell physiology along with a temporal function of leptin receptor-induced mTORC1 activity in repression of Treg proliferation68,69. This role of leptin in mTOR regulation, along with earlier evidence of its capacity to enhance interferon-gamma (IFN-γ) production from cultured CD4 T cells70, is intriguing in the context of metabolism of the patient with cancer. Another important aspect of mTOR regulation is suggested from the combination of recent and older work on signaling by Notch receptors. In addition to a requirement for Notch in thymic T-cell development, a variety of articles have linked this receptor to regulation of the balance of effector cell subset formation, including evidence for Notch-stimulated mTOR controlling Treg71–82. Of note, Notch stimulates both mTORC1 and mTORC2 activity; each of these branches is important for T helper subset specification, including distinct functions of mTORC1 and mTORC2 in Treg53,82–85, and is needed for Notch function in thymocytes84,85. PI3K-mTOR activity initiated by the inducible co-stimulator ICOS is yet another driver of mature T-cell differentiation (e.g. the follicular helper subset, TFH)40,86.\n\n\nSo, what could be the m(a)TOR with the beautiful picture?\n\nNotwithstanding the beautiful picture, the PI3K-mTOR signaling pathway further exemplifies some pitfalls of relying on a binary style of drawing conclusions and the previously noted complications in conceiving of T-cell differentiation independent from the dynamism of time and space (hence, micro-environment). At present, the dominant experimental tool consists of stable, nearly complete losses of function. This suggests a need for caution about physiological perturbation-response relationships (Figure 2). This concern is strengthened by evidence that the magnitude and duration of mTOR kinase activation vary according to the circumstances of stimulation (including the mix and timing of cytokine exposures after initial activation) and work showing that the effects of rapamycin depend on the strength and nature of stimulation87,88. The preceding section suggests yet another gap in our approaches and understanding. As ever more phenomena are attributed to the activation of mTOR or its nearly complete failure — e.g. 89,90 — in addition to previously cited work (e.g. 39,40,61,62,65,66,69), it will be intriguing to sort out mechanisms; already, well over a half-dozen disparate receptors with very different ligands are each reportedly necessary for approximately 80% (or more) of the activity on a branch of the mTOR pathway.\n\nAs highlighted by evolving single-cell stimulus-response research34,35, a given stimulus will often yield a probability of evoked response well below 1, and this stimulus-probability curve will vary within a series of gene expression or downstream signal intensities pertinent to T-cell differentiation or function. Accordingly, as cell conditions or pre-existing programs vary or as different extents of impairment to a given signal relay are imposed, the responses will exhibit variegation among cells in a population. Similar principles are postulated almost certainly to apply to the time element (e.g. how long does it take for a condition to change histone post-translational modifications or epigenetic modification of DNA or to change the level of expression of a target gene?). As noted in Figure 4, local conditions vary even within a single tumor mass (setting aside known differences between metastatic and primary tumors).\n\nThere might be some super-complex requiring all these disparate elements at once. Also, there may prove to be subtle variations in the time course or protocols even as the nature of developing scientific stories emphasizes “optimizing conditions” to maximize a particular observation. On a mechanistic note, one potential resolution of the conundrum is whether there are relay or cross-talk effects (e.g. whether mTOR activity stimulated by one receptor depends on antecedent induction by a different stimulus). This model may apply in the case of Notch-stimulated mTOR, which then feeds into “tuning” TCR sensitivity73, a model separately proposed for the transcription factor nuclear factor-kappa-B (NF-κB) in thymocytes91. Another possibility links to changes in subcellular localization. Key kinases in this pathway partition among compartments (for instance, AKT and the mTORC1 target S6K)92–95. In the case of FoxO phosphorylation, it is appealing to consider that nuclear AKT is a crucial element of the equation. Of note, the efficiency of nuclear sampling for phosphorylated forms of AKT varies dramatically among means of cell extraction (an analytic problem that is even worse with intracellular staining for flow cytometry). So, there may be differential sampling of these compartments (e.g. nucleus versus cytosol) in various articles. Among the mutually compatible possibilities is that function is effected in large part via relays and supra-molecular complexes akin to signaling involving MAPK or NF-κB activation96–99.\n\nSecond, important work on FoxO1 and stability of immune homeostasis mediated by Treg used a knock-in that permitted quantitation of the extent to which TCR stimulation could evict Foxo1 from the nucleus. In this analysis, two key findings were that (i) for FoxP3+ (Treg) and FoxP3− (Tconv) cells, signal intensities for both Erk and the mTOR–AKT–FoxO1 were quite different, as was the extent of FoxO1 redistribution, and (ii) at best, partial nuclear exclusion was driven when focusing on Treg59. Accordingly, even haplo-insufficiency for FoxO1 may overestimate the extreme end of a dynamic range that is achieved in vivo. An intriguing side point pertaining to transforming growth factor-beta-rich tumor environments is that this cytokine activates mTOR and AKT100–102 even though AKT is supposed to suppress Treg fate or function. In light of the evidence that signal distribution and “interpretation” within Treg can differ dramatically from those in the conventional CD4+ T cell, detailed analysis of how PD-1 actually affects mTORC1, mTORC2, and other signaling pathways is needed. More broadly, there is a need in the field to seem less absolute and oversimplistic. In addition to the factors already noted, recent work in a developmental system provides strong evidence that the use of longer-term and complete gene inactivation or loss of function can elicit compensatory circuits that are avoided when a signal is attenuated more acutely with RNA interference (RNAi)103. As one working example, disparate articles even within the spheres of both CTL and conventional helper T-cell differentiation observe quite different outcomes (both qualitatively and quantitatively) with perturbing the same pathway53,79–81,83,104–107 (Figure 3). These differences probably represent combinations of fine gradations in the timing and extent of loss-of-function perturbations and hence distinct points on uncharacterized dose-response curves, along with variance among models and, who knows, maybe even microbiomes.\n\n(A) An oversimplified view segregating functions of mammalian (or mechanistic) target of rapamycin (mTOR) complexes with distinct functions mapping one on one with T helper (Th) subsets. (B) A partial integration of more complete information as represented in the cited articles51–53,57–59,79–83 and related work. Notable differences include that, presumably as a function of differences in conditions, the literature supports roles for each mTOR complex in promoting each of the peripherally acquired CD4 effector phenotypes (Th1, Th2, Th17), while the effects on regulatory T (Treg) are complex, with mTOR and mTORC2 shown to restrain induction of FoxP352,53 but mTORC1 vital for the suppressive function of these cells83. If non-drastic decreases in nuclear FoxO1 are assumed to attenuate Treg function59, then enhanced mTORC2 activity in Treg might decrease their inhibitory properties. The connection of mTORC2 to Th17 function is inferred from mTORC2 activation of SGK1 and the function of this latter factor in promoting pathogenic Th1746. Abbreviations: IFN-γ, interferon-gamma; IL, interleukin; RA, retinoic acid; TCR, T-cell receptor; TGF-β, transforming growth factor-beta.\n\nThird, T cells are quite the moving target. Intra-vital imaging, albeit with caveats relating to the potential impact of clonal frequency on responses108,109, shows that after a period of arrest, T cells resume roaming within the lymphoid organ. Moreover, activated T cells leave the lymphoid organs to circulate and be recruited to tissues – especially if there are sites of inflammation. Further complexities related to inflammation and the kaleidoscope of the micro-environment include hypoxia, angiogenesis, and tumor-associated myeloid-lineage cells. The metabolic landscape differs among the arterial, venous, and lymphatic circulations as well as the tissues, which in turn will be different if challenged by metastatic cancer cells or microbial invasion (e.g. intracellular pathogens) (Figure 4). Co-stimulation and hypoxia response mechanisms impact motility, mTOR activities, and T-cell phenotypes, although sometimes with opposite experimental results that may depend on the metabolic environment of the tested cells, the lymphocyte subset, whether the T cell had previously been activated, and the form of stimulus (e.g. cytokine)110–115. As a tumor grows, mutability can yield “self” peptides of altered sequence that may then be perceived as foreign (“non-self”)116. Indeed, new evidence suggests a correlation or even mechanistic connection between the degree to which such epitopes are generated and the responsiveness to checkpoint inhibitors117,118. However, the universe of peptides that activate Treg may differ from those that engage TCR on conventional CD4 and CD8 T cells, and MHC-neopeptide complexes will include antagonists that drive clonal anergy119.\n\nShown is a simplified overview of the multiple and diverse sites at which each type of T cell may encounter signals and tumor-derived antigens during growth and metastasis for carcinomas, together with a diagram underscoring likely variations of malignant cells within the tumor mass itself. In addition to action within the tumor itself, which may not be a site of initial tumor antigen presentation to and activation of T cells, either live or dead tumor cells, or antigens derived from them, traffic and flow to secondary lymphoid organs (spleen; lymph node), in either particulate or cell-associated (e.g. within a dendritic or movable phagocytic cell) form. APC, antigen-presenting cell; Treg, regulatory T.\n\n\nImagining parts of a future\n\nTo recap, an attractive model based on “PI3K–AKT–mTOR” signals that drastically alter nuclear FoxO protein levels could link checkpoint therapies to changes in immune responsiveness controlled by Treg. However, constrained by the state of current technologies, the first-wave studies are based on relatively extreme loss- or gain-of-function changes that are fixed and static. In the worst instances, inferences are drawn and conclusions are strongly stated based on knockouts that yield starting T-cell populations identifiably different from the reference “wild-type controls”. But even setting aside this problem, temperance and caution are called for in making a dogma from reasonable inferences based on immutable perturbations in systems that have clear feedback inhibition and counter-regulation. Moreover, evidence is lacking as to where key changes in the relevant cells occur – with the cells in question highly dynamic. It is likely a priori that shifts in the environment as activated T cells move within and out from tissues modulate the probabilities of particular fates (Figure 4).\n\nLooking ahead, then, it is suggested that the list of needs includes the following:\n\n1. Cultural change in science, with fewer oversimplifications that rely on overly categorical divisions between function of A versus function of B (Figure 3). A central challenge stems from a combination of the overly definitive presentation of findings along with an implicit expectation of relatively universal “explanations” and an undue degree of “buy-in” to static conceptual schemata (explanatory cartoons).\n\n2. More information on heterozygote phenotypes for loss-of-function perturbations of these pathways rather than focusing exclusively on the extremes – more broadly, better and more widespread attention to and precision with dose-response curves and understanding of stochastic variance (Figure 2).\n\n3. Development and use of “tools” and methodologies whereby graded changes in activity of the systems can be imposed – ideally, for more limited periods of time even than a laudable model making use of 4-OH-tamoxifen-activated estrogen receptor ligand-binding domains120. Application of optogenetic tools or their marriage to new genetics may prove valuable in moving toward this goal, ideally with the added value of permitting spatially restricted activation121–124.\n\n4. More ready supply of existing tools, along with development of better means for analyzing the actual pool of fuels (e.g. hexoses, glutamine, fatty acids, and oxygen) and metabolites in the interstitial spaces which are the soils among which cells are moving, along with\n\n5. A capacity to follow the cells and better detect their signaling in situ124–126 across the timescales relevant to their development and functions.", "appendix": "Competing interests\n\n\n\nThe author declares that he has 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\nCouzin-Frankel J: Breakthrough of the year 2013. Cancer immunotherapy. Science. 2013; 342(6165): 1432–3. PubMed Abstract \| Publisher Full Text\n\nTopalian SL, Hodi FS, Brahmer JR, et al.: Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med. 2012; 366(26): 2443–54. 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PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nGuntas G, Hallett RA, Zimmerman SP, et al.: Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins. Proc Natl Acad Sci U S A. 2015; 112(1): 112–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVictora GD, Schwickert TA, Fooksman DR, et al.: Germinal center dynamics revealed by multiphoton microscopy with a photoactivatable fluorescent reporter. Cell. 2010; 143(4): 592–605. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nMüller AJ, Filipe-Santos O, Eberl G, et al.: CD4+ T cells rely on a cytokine gradient to control intracellular pathogens beyond sites of antigen presentation. Immunity. 2012; 37(1): 147–57. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nMoreau HD, Bousso P: Visualizing how T cells collect activation signals in vivo. Curr Opin Immunol. 2014; 26: 56–62. PubMed Abstract \| Publisher Full Text" }	[ { "id": "12542", "date": "18 Feb 2016", "name": "Protul Shrikant", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12543", "date": "18 Feb 2016", "name": "Michael Dustin", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12544", "date": "18 Feb 2016", "name": "Anjana Rao", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12545", "date": "18 Feb 2016", "name": "Hongbo Chi", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-191
https://f1000research.com/articles/5-189/v1	18 Feb 16	{ "type": "Software Tool Article", "title": "FreeSASA: An open source C library for solvent accessible surface area calculations", "authors": [ "Simon Mitternacht" ], "abstract": "Calculating solvent accessible surface areas (SASA) is a run-of-the-mill calculation in structural biology. Although there are many programs available for this calculation, there are no free-standing, open-source tools designed for easy tool-chain integration. FreeSASA is an open source C library for SASA calculations that provides both command-line and Python interfaces in addition to its C API. The library implements both Lee and Richards’ and Shrake and Rupley’s approximations, and is highly configurable to allow the user to control molecular parameters, accuracy and output granularity. It only depends on standard C libraries and should therefore be easy to compile and install on any platform. The library is well-documented, stable and efficient. The command-line interface can easily replace closed source legacy programs, with comparable or better accuracy and speed, and with some added functionality.", "keywords": [ "Structural Bioinformatics", "Solvent Accessible Surface Area", "Command-line tool", "C library", "Python module" ], "content": "Introduction\n\nExposing apolar molecules to water is highly unfavorable, and minimizing the hydrophobic free energy is an important driving force in the folding of macromolecules (Finkelstein & Ptitsyn, 2002). The solvent accessible surface area (SASA) of a molecule gives a measure of the contact area between molecule and solvent. Although the exact quantitative relation between surface area and free energy is elusive, the SASA can be used to compare different molecules or different conformations of the same molecule, or for example measure the surface that is buried due to oligomerization.\n\nTo define the SASA, let a spherical probe represent a solvent molecule. Roll the probe over the surface of a larger molecule. The surface area traced by the center of the probe is the SASA of the larger molecule (Lee & Richards, 1971). Two classical approximations are commonly used to calculate SASA: one by Lee and Richards (L&R) where the surface is approximated by the outline of a set of slices (1971), and one by Shrake & Rupley (1973) (S&R) where the surface of each sphere is approximated by a set of test points. The SASA can be calculated to arbitrary precision by refining the resolution of both. The surface area can also be calculated analytically (Fraczkiewicz & Braun, 1998), which is useful when the gradient is needed, or by various other approximations, tailored for different purposes (Cavallo et al., 2003; Drechsel et al., 2014; Sanner et al., 1996; Weiser et al., 1999; Xu & Zhang, 2009).\n\nThere are many tools available to calculate SASA. The most popular program for command line use is probably NACCESS (Hubbard & Thornton, 1993) (freely available for academic use), which is an efficient Fortran implementation of the L&R approximation. Another well-known command line tool is DSSP, which primarily calculates the secondary structure and hydrogen bonds of a protein structure, but provides the SASA as well (Touw et al., 2015) (using S&R, open source). There are also some web services available, for example Getarea, which calculates the surface analytically (Fraczkiewicz & Braun, 1998), and Triforce which uses a semianalytical tessellation approach (Drechsel et al., 2014) (also available for command line use). In addition, most molecular dynamics simulation packages include tools to analyze SASA from trajectories.\n\nFreeSASA is intended to fill the same niche as NACCESS, and a number of other similar programs: a simple and fast command-line tool that “does one thing and does it well” and can be easily integrated into tool chains. The advantage of FreeSASA is that it is open source (GNU General Public License 3), and provides both C and Python APIs in addition to its command line interface. It has sensible default parameters and no obligatory configuration for casual users (the only required input is a structure), but also allows full control over all calculation parameters. Dependencies have been kept to a minimum: compilation only requires standard C and GNU libraries. The library is thread-safe, and some effort has gone into dealing gracefully with various errors. The code ships with thorough documentation, which is also available online at http://freesasa.github.io/doxygen/. Although functionality and availability have been the primary motivating factors for writing this library, performance tests show that FreeSASA is as fast as or faster than legacy programs when run on a single CPU core. In addition, a large portion of the calculation has been parallelized, which gives significant additional speed when run on multicore processors.\n\n\nMethods\n\nCalculations. Both S&R and L&R are pretty straightforward to implement, and both require first determining which atoms are in contact, and then calculating the overlap between each atom and its neighbors. Finding contacts is done using cell lists, which means the contact calculation is an O(N) operation. Both algorithms then treat each atom independently, making also the second part of the calculation O(N). In addition, this second part is trivially parallelizable.\n\nFor L&R, instead of slicing the whole protein in one go, each atom is sliced individually. The L&R calculation is thus parameterized by the number of slices per atom, i.e. small atoms have thinner slices than large atoms.\n\nThe Fibonacci spiral gives a good approximation to an even distribution of points on the sphere (Swinbank & Purser, 2006), allowing efficient generation of an arbitrary number of S&R test points. The cell lists provide the first of the two lattices in the double cubic lattice optimization for this algorithm (Eisenhaber et al., 1995), the second lattice (for the test points) is not implemented in FreeSASA, for now.\n\nThe correctness of the implementations was tested by first inspecting the surfaces visually. In the two atom case, results were verified against analytical calculations. Another verification came from comparing the results of high precision SASA calculations using the two independent algorithms. In addition, using the L&R algorithm gives identical results to NACCESS when the same resolution and atomic radii are used.\n\nRadius assignment. An important step of the calculation is assigning a radius to each atom. The default in FreeSASA is to use the ProtOr radii by Tsai et al. (1999). The library recognizes the 20 standard amino acids (plus Sec and Pyl), and the standard nucleotides (plus a few nonstandard ones). Tsai et al. do not mention phosphorus and selenium; these atoms are assigned a radius of 1.8 and 1.9 Å respectively.\n\nBy default, hydrogen atoms and HETATM records are ignored in Protein Data Bank (PDB) files. If included, the library recognizes three common HETATM entries: the acetyl and NH2 capping groups, and water, and assigns ProtOr radii to these. Otherwise the van der Waals radius of the element is used, taken from the paper by Mantina et al. (2009). For elements outside of the 44 main group elements treated by Mantina et al., or if completely different radii are desired, users can provide their own configuration.\n\nUsers can specify their own atomic radii either through the API or by providing a configuration file. The library ships with a few sample configuration files, including one that provides a subset of the NACCESS parameterization, and one with the default ProtOr parameters. In addition, scripts are provided to automatically generate ProtOr configurations from PDB CONECT entries, such as those in the Chemical Component Dictionary (Westbrook et al., 2015). These can then be appended to the default configuration.\n\nBuilding the FreeSASA library and command-line interface only requires standard C and GNU libraries and a C99-compliant compiler, and should be straightforward on any UNIX system (has been tested in Mac OS X 10.8 and Debian 8), and not too difficult in Windows (not tested). Building the Python bindings requires Cython (tested with version 0.21). The library ships with an Autotools build configuration, but the source itself is simple enough to be possible to compile “manually”, if necessary.\n\nCommand-line interface. Building FreeSASA creates the binary freesasa. The simplest program call, with default parameters, is\n\n\n\nusing the structure with PDB code 3wbm as an example (a protein-RNA complex). The above produces the following output\n\n\n\n\n\n\n\n\n\nThe numbers in the results section are the SASA values (in Å2) for the respective groups of atoms.\n\nAs an illustration of a few of the other configuration options, and how to use the program as a PDB file filter, the command\n\n\n\ncalculates the SASA of a PDB-file passed via stdin, using 100 slices per atom. The flag --no-log suppresses the regular output. The output will instead, because of the flag --print-as-B-values, be the provided PDB-file with the SASA of each atom replacing the temperature factors, and the atomic radii stored in the occupancy factor field.\n\nBy calling with the option --chain-groups,\n\n\n\ntwo calculations are appended to the original output, one where only the four chains A, B, C and D have been included, and one with only X and Y.\n\nThe option --select can be used to select a set of atoms using a subset of the selection syntax used in the program Pymol (DeLano, 2002). For example, the command\n\n\n\nwill produce the following output (after the regular output shown above)\n\n\n\nwhere RNA is simply the user-defined name of the selection, and the number the contribution to the total SASA from the bases A, U, G and C (which we in this particular case could have got by simply adding the areas for the chains X and Y in the sample output above).\n\nThe command\n\n\n\nprints a help message listing all available options, including other ways to redirect output and how to change different calculation parameters (the most detailed information can be found online at http://freesasa.github.io/doxygen/CLI.html).\n\nC API. The C code below illustrates how to perform a SASA-calculation on the same PDB-file as above, using the C API, with default parameters. The functions and types used are all defined in the header freesasa.h.\n\n\n\nThe two points where null pointers are passed as arguments are places where atom classifiers and calculation parameters could have been provided. A more elaborate example that includes error handling and freeing of allocated resources can be seen in Figure 1.\n\nIllustration of how to use the C API including rudimentary error handling.\n\nThe API also allows the user to calculate the SASA of a set of coordinates with associated radii. The code below puts two atoms at positions x→1= (1, 1, 1) and x→2= (2, 2, 2) with radii r1 = 2 and r2 = 3, respectively, and outputs the SASA of the individual atoms.\n\n\n\nPython API. The library includes Python bindings that export most of the C API to Python. The Python code below gives the same output as the example in Figure 1. Error handling is excluded for brevity.\n\n\n\n\n\n\n\n\nResults\n\nThe computational efficiency of the two algorithms was compared by running the FreeSASA command-line program with different parameters on a set of 88 PDB structures selected from the PISCES database (Wang & Dunbrack, 2003) (see Dataset 1 for a list). PISCES specifies a specific chain in each structure, but in the following all chains were used, which resulted in the largest structure having over 30,000 atoms (1jz8). To average out some variation in the running time in these rather short calculations (in some cases < 10 ms), the fastest calculations were run two to five times. As we will see below, error bars are relatively small along that axis.\n\nTo measure the accuracy of the two algorithms, a reference SASA value, Aref, was calculated using L&R with 1000 slices per atom for each structure. The error of a given SASA-value, A, is then ε = \|A–Aref\|/N, where N is the number of atoms in the structure. Calculation time T is measured as the wall time of the entire calculation including reading and writing files. Dataset 2 contains the values of A, Aref, N and T used to produce Figure 2 and Figure 3.\n\nThe mean of the error ε in SASA vs T/N, for the two algorithms in FreeSASA plus the programs NACCESS and POPS. Labels indicate the resolution used for each set of calculations, and error bars the standard error along both axes. The solid lines are only there to guide the eye, and the dashed lines indicate the analogous lines when using 2 and 4 threads in FreeSASA. An L&R run with 1000 slices was used as Aref when calculating ε for both approximations. NACCESS uses L&R and was run with three values of the z-parameter (0.1, 0.05 and 0.01, corresponding to 10, 20 and 100 slices per atom), a run with z-parameter 0.005 was used as Aref (using even lower z-values gave inconsistent results). The NACCESS reference calculation was also used as reference for POPS. All programs were compiled using GCC 4.9.3 with the optimization flag “-Ofast” and the tests were run on an Intel Core i5-2415M CPU at 2.30 GHz. The raw data for this figure can be found in Dataset 2.\n\nThe histograms shows the distribution of the calculation time using two or four threads divided by the time using one thread. Thus if this fraction is two or four, respectively, we have “perfect” parallelization. The legends indicate the resolution of the calculation: for L&R, slices per atom, and for S&R, number of test points. The raw data for this figure can be found in Dataset 2.\n\nFigure 2 shows ε versus T/N, averaged over the 88 PDB structures. At low resolution S&R is considerably faster than L&R, and at high resolution L&R is faster, with a crossover at around 1000 test points or 20 slices per atom (20 slices is the default setting in FreeSASA).\n\nComparisons were done with NACCESS 2.1.1 (Hubbard & Thornton, 1993), DSSP 2.2.1 (Touw et al., 2015), NSOL 1.7 (Masuya, 2003), POPS 1.6.4 (Cavallo et al., 2003) and Triforce 0.1 (Drechsel et al., 2014). The list could potentially have been a lot longer; some programs were left out on the basis of being closed source, poorly documented or no longer available. NACCESS was included in spite of its limiting license due to its popularity. The SASA facilities in molecular dynamics packages were not considered since these cater to a different use case.\n\nNACCESS allows the user to choose arbitrary resolution and can therefore be used as a reference for itself, and POPS was optimized with NACCESS as reference. NACCESS uses L&R and performs very similarly to FreeSASA using L&R. The POPS algorithm is intended as a fast coarse-grained approximation; its authors state an average error of 2.6 Å2 per atom (Cavallo et al., 2003). In Figure 2 the mean ε is lower than that, which is expected, since this error is measured over the total SASA, not atom by atom. A fit showed that POPS runs in O(N2) time (using the data in the file pops.dat in Dataset 2), which to some extent explains the relatively long mean calculation time per atom.\n\nThe other programs listed above were left out of Figure 2 because they can not be compared under the same premises. DSSP calculates many different things in addition to its 200 test-point S&R-calculation, and the total running time is therefore naturally longer than the corresponding calculation in FreeSASA, although the accuracy should be comparable for the same number of test points. The program NSOL uses S&R, but does five different SASA calculations on the same input using different parameters. The NSOL code was modified to only do one of the five calculations, and is then only slightly slower than FreeSASA using the same number of test points. Lastly, Triforce is not suitable for comparison in this particular use case because it has a high initialization cost, which makes it slow for calculating the SASA of an isolated structure.\n\nIn single-threaded mode, FreeSASA using L&R is almost indistinguishable from NACCESS in Figure 2, but it is significantly faster when 2 or 4 threads are used. The effect of spreading the calculation over several threads is shown in more detail in Figure 3. Since the generation of cell lists is not parallelized, using more than one thread only gives a significant performance benefit in the high resolution limit. Based on these results, the default has been set to two threads. Depending on the nature of the calculations, this speedup can make a noticeable difference.\n\n\nSummary\n\nFreeSASA is an efficient library for calculating the SASA of protein, RNA and DNA structures. Its main advantages over other commonly used tools is that it is open source, easily configurable and can be used both as a command line tool, a C library and a Python module. The tests above demonstrate that it runs as fast as, or faster than, some popular tools at a given resolution, and can be boosted further by parallelizing the calculation.\n\n\nData and software availability\n\nF1000Research: Dataset 1. List of PDB codes used for the performance analysis, 10.5256/f1000research.7931.d112977 (Mitternacht, 2016a).\n\nF1000Research: Dataset 2. Zip-archive with raw data for the performance analysis in Figure 2 and Figure 3, 10.5256/f1000research.7931.d112978 (Mitternacht, 2016b).\n\nLatest source code and software available from http://freesasa.github.io/\n\nArchived source code as at the time of publication http://dx.doi.org/10.5281/zenodo.45239 (Mitternacht, 2016c).\n\nLicense GNU GPL v3 (http://www.gnu.org/licenses/gpl-3.0.en.html).", "appendix": "Competing interests\n\n\n\nNo competing interests are disclosed.\n\n\nGrant information\n\nThe author declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nThanks go to Edvin Fuglebakk for comments on the code and documentation. Thanks to Sandhya P. Tiwari and Anders Irbäck for comments on the manuscript. Thanks to João Rodrigues for suggestions for improved functionality.\n\n\nReferences\n\nCavallo L, Kleinjung J, Fraternali F: POPS: A fast algorithm for solvent accessible surface areas at atomic and residue level. Nucleic Acids Res. 2003; 31(13): 3364–3366. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDeLano WL: The PyMOL molecular graphics system. 2002. Reference Source\n\nDrechsel NJ, Fennell CJ, Dill KA, et al.: TRIFORCE: Tessellated Semianalytical Solvent Exposed Surface Areas and Derivatives. J Chem Theory Comput. 2014; 10(9): 4121–4132. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEisenhaber F, Lijnzaad P, Argos P, et al.: The double cubic lattice method: efficient approaches to numerical integration of surface area and volume and to dot surface contouring of molecular assemblies. J Comput Chem. 1995; 16(3): 273–284. Publisher Full Text\n\nFinkelstein AV, Ptitsyn O: Protein physics: a course of lectures. Academic Press, London, 2002. Reference Source\n\nFraczkiewicz R, Braun W: Exact and efficient analytical calculation of the accessible surface areas and their gradients for macromolecules. J Comput Chem. 1998; 19(3): 319–333. Publisher Full Text\n\nHubbard SJ, Thornton JM: NACCESS. Computer Program, Department of Biochemistry and Molecular Biology, University College London, 1993. Reference Source\n\nLee B, Richards FM: The interpretation of protein structures: estimation of static accessibility. J Mol Biol. 1971; 55(3): 379–400. PubMed Abstract \| Publisher Full Text\n\nMantina M, Chamberlin AC, Valero R, et al.: Consistent van der Waals radii for the whole main group. J Phys Chem A. 2009; 113(19): 5806–5812. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMasuya M: NSOL: A numerical calculation program of molecular surface area, volume, and solvation energy. 2003. Reference Source\n\nMitternacht S: Dataset 1 in: FreeSASA: An open source C library for solvent accessible surface area calculations. F1000Research. 2016a. Data Source\n\nMitternacht S: Dataset 2 in: FreeSASA: An open source C library for solvent accessible surface area calculations. F1000Research. 2016b. Data Source\n\nMitternacht S: FreeSASA 1.0.1: Solvent Accessible Surface Area Calculations. Zenodo. 2016c. Data Source\n\nSanner MF, Olson AJ, Spehner JC: Reduced surface: an efficient way to compute molecular surfaces. Biopolymers. 1996; 38(3): 305–320. PubMed Abstract \| Publisher Full Text\n\nShrake A, Rupley JA: Environment and exposure to solvent of protein atoms. Lysozyme and insulin. J Mol Biol. 1973; 79(2): 351–371. PubMed Abstract \| Publisher Full Text\n\nSwinbank R, Purser RJ: Fibonacci grids: A novel approach to global modelling. Q J R Meteorol Soc. 2006; 132(619): 1769–1793. Publisher Full Text\n\nTouw WG, Baakman C, Black J, et al.: A series of PDB-related databanks for everyday needs. Nucleic Acids Res. 2015; 43(Database issue): D364–D368. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTsai J, Taylor R, Chothia C, et al.: The packing density in proteins: standard radii and volumes. J Mol Biol. 1999; 290(1): 253–266. PubMed Abstract \| Publisher Full Text\n\nWang G, Dunbrack RL Jr: PISCES: a protein sequence culling server. Bioinformatics. 2003; 19(12): 1589–1591. PubMed Abstract \| Publisher Full Text\n\nWeiser J, Shenkin PS, Still WC: Approximate atomic surfaces from linear combinations of pairwise overlaps (LCPO). J Comput Chem. 1999; 20(2): 217–230. Publisher Full Text\n\nWestbrook JD, Shao C, Feng Z, et al.: The chemical component dictionary: complete descriptions of constituent molecules in experimentally determined 3D macromolecules in the Protein Data Bank. Bioinformatics. 2015; 31(8): 1274–1278. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nXu D, Zhang Y: Generating triangulated macromolecular surfaces by Euclidean Distance Transform. PLoS One. 2009; 4(12): e8140. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "12524", "date": "26 Feb 2016", "name": "Simon J. Hubbard", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a simple and effective implementation of the two main heuristics used for calculating solvent accessible surface area of atomic structures such as proteins. The algortihm, FreeSASA, is suitably described and tested in the article. I see no real need to ask for any changes, as the author has done a very professional and nice job here. It downloads and installs very smoothly and offers most of the functionality that NACCESS offers, so will be appreciated by legacy users I would imagine (though some things have not been implemented, probably with good reason - either way, not a problem for me - and the author can react to requests or users can do a little work to get what they want in terms of formatting). I am sure the ability to thread across multiple cores will be beneficial and speed things up, and the API will make it slot into python pipelines etc. It's possible that it won't directly supplant some NACCESS dependencies which rely on its rather old formatting (.rsa file for example perhaps) but this is probably only a minor concern and easily fixed if needed (since its fully open source, its probably reduced to changing a few print statements anyway).", "responses": [] }, { "id": "12528", "date": "29 Feb 2016", "name": "Yaoqi Zhou", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSolvent accessible surface area is a frequently calculated quantity in structural bioinformatics. Although many tools are available, an open source, easy-to-use program is certainly welcome. The author did a careful test and performed comparative studies to existing tools. The code is clearly written and well documented. There are no major changes required for this clearly written manuscript. Here are some minor questions requiring further clarifications and additions.For multiple chains or a protein-ligand complex structure, does Freesasa yield the ASA for isolated chains or chains in the complex structure? It would be better if the program has an option to calculate the change of ASA on a residue before and after binding to another molecule (ligand, RNA, DNA, another chain) assuming that there is no structural change upon binding. This would allow to identify the functional residues. Please clarify the command to produce a residue-level ASA, rather than the atomic level ASA. What are default recommendations for the ASA calculation in term of resolution required or choice of LR and SR approximations? Are the default atomic radii employed here the same as used in DSSP and/or NACCESS? If different, what are the main differences? Is this method faster than analytical methods? If so, by how much? Can authors provide a table that lists all possible commands? The program would be more useful if it can directly read in Biopython data structure and work with pymol.", "responses": [ { "c_id": "1840", "date": "29 Feb 2016", "name": "Simon Mitternacht", "role": "Author Response", "response": "Thanks for your comments and suggestions. I interpret most of them as being better solved by improving the functionality and/or documentation of the program itself, rather than revising the paper. For the sake of brevity not all the functionality is described in the paper, but is available in the online documentation.The default behavior is to calculate the SASA of the chains in the complex. The command option '--chain-groups' described in the paper allows the user to look at combinations of chains in isolation. The option '--separate-chains', is not discussed in the paper, but can be used to treats each chain separately (i.e. for a four chain protein '--chain-groups=A+B+C+D' would be equivalent to '--separate-chains'). My intention was to give the user the ability to calculate the total ASA of different combinations of chains, and then do the arithmetic of the changes themselves. I will look into adding command-line options to do this automatically in future versions of the program. The option '--foreach-residue' (not described in the paper) can be used to print the SASA of each residue. It is hard to give a general recommendation here, but I chose to use the same default resolution as NACCESS since this seems to be an accepted standard. If one is looking for changes due to minor conformational changes, a higher resolution might be needed. The average of Aref/N in Dataset 2 is 5.6 Å2, and the average error in Figure 2 for the default resolution is around 0.002 Å2, i.e. the error in the total SASA (A) is on average 0.002/5.6 < 1/1000. As mentioned in the paper, the ProtOr atomic radii are used. I think the easiest way of comparing these with those employed in NACCESS and DSSP is to compare the radii in the configuration-files supplied with the program (in the directory share/). I was not able to get hold of FANTOM or any other analytical program, so I have unfortunately not been able to compare directly. As mentioned I compared with the semi-analytical Triforce, which was significantly slower when run on a single structure due to high initialization cost. I assume Triforce performs better when the same instance of the program is run on many structures, but that is a different use case. The command 'freesasa -h' lists all options. I decided not to provide this list in the paper because it will probably expand as time goes, and then the paper would quickly become outdated. Thank you for these suggestions, I will look into ways of adding more functionality to the Python bindings." } ] } ]	1	https://f1000research.com/articles/5-189
https://f1000research.com/articles/5-186/v1	17 Feb 16	{ "type": "Review", "title": "Recent advances in the understanding of endometriosis: the role of inflammatory mediators in disease pathogenesis and treatment", "authors": [ "Warren Nothnick", "Zahraa Alali", "Zahraa Alali" ], "abstract": "In this review, we focus on recent advancements in our understanding of the roles of inflammatory mediators in endometriosis pathophysiology and the potential for improved therapies based upon targeting these pathways. We review the association between endometriosis and inflammation and the initial promise of anti-tumor necrosis factor therapies based upon experimental evidence, and how and why these studies have not translated to the clinic. We then discuss emerging data on the role of inter-relationship among macrophage migration inhibitory factor, prostaglandin E2, and estrogen receptor-beta, and the potential utility of targeting these factors in endometriosis treatment. In doing so, we highlight the strengths and discuss the current research on identification of novel, anti-inflammatory-based therapy and the necessity to expand experimental endpoints to include clinically relevant measures when assessing the efficacy of potential new therapies for endometriosis.", "keywords": [ "endometriosis", "prostaglandin E2", "estrogen receptor-beta" ], "content": "Introduction\n\nIn this short report, we focus on recent advances in our understanding on the role of inflammatory mediators in the pathophysiology of endometriosis and the potential utility of therapeutic agents that target their action. We discuss how initial studies of related targets, such as tumor necrosis factor-alpha (TNF-α), failed to result in novel, non-hormonal therapy, and we introduce new players which have gained attention for their role in the pathophysiology of endometriosis. We discuss emerging data on the role and inter-relationship among macrophage migration inhibitory factor (MIF), prostaglandin E2 (PGE2), and estrogen receptors alpha (ER-α) and beta (ER-β), and the potential utility of targeting these factors in endometriosis treatment. We highlight the strengths of current research on the identification of novel, anti-inflammatory-based therapy and discuss the necessity to expand experimental endpoints to include clinically relevant measures when assessing the efficacy of potential new therapies for endometriosis.\n\n\nEndometriosis and limitations with current therapies\n\nEndometriosis is a disease which affects women of reproductive age and is defined as the growth of endometrial tissue in ectopic locations, primarily within the pelvic cavity. Endometriosis is a chronic disease characterized by pelvic pain and infertility, and affects over 70 million women worldwide. Given a 10% prevalence rate among women of reproductive age, the annual costs of endometriosis were estimated at $22 billion in 2002 in the US alone. These costs are considerably higher than those related to migraine or Crohn’s disease1. One reason for the high cost is that there are insufficient treatments for the disease. Current medical treatment approaches rely on the fact that endometriosis is an estrogen-dependent disease. Yet, relief is at the expense of induction of a hypo-estrogenic state, which is counterproductive for infertility treatment and associated with unwanted menopausal-like side effects, the major drawback being a potential reduction in bone density.\n\nThese drawbacks of current endometriosis treatments often lead to abandonment of medical therapy and repeated surgical therapy. As such, there is a great need for the identification of novel targets for endometriosis treatment. Treatments that directly target the endometriotic implant would overcome the abovementioned shortcomings. Unfortunately, there are no current endometriotic implant-specific treatment options that have been shown to be successful or act independently of steroid production or action. The potential of targeting inflammatory mediators associated with endometriosis has been intensely investigated over the past three decades with varying degrees of success. The purpose of the following review is to provide a brief history of how these targets emerged as potential, non-hormonal-based therapies, outline what we learned from prior studies, and discuss why early therapeutics have yet to show efficacy. From there, we highlight the study of emerging mediators of inflammation associated with endometriosis and define the cooperative interaction among these mediators in the pathophysiology of endometriosis, as well as discuss the future application of targeting these mediators toward development of novel, anti-inflammatory therapeutic agents for endometriosis treatment.\n\n\nEndometriosis, inflammation, and progesterone resistance\n\nIt has long been acknowledged by both researchers and clinicians that endometriosis is a disease associated with inflammation and elevated cytokine levels2,3. Altered cytokine production by both cells of the immune system and the endometriotic lesion tissue has been proposed (discussed below for each of the specified cytokines) to contribute to these elevated cytokine levels. One of the driving factors for the enhanced production of endometriotic lesion cytokines is an altered progesterone responsiveness associated with the disease. Progesterone exhibits anti-inflammatory actions, and as such, progesterone analogs have been used to treat endometriosis and its associated symptoms4. Progestin (progesterone) treatment appears to be successful in most5, but not all6, women, and not all progestin formulations are effective in reducing endometriosis-associated pain7. This inconsistency could be due to the progesterone resistance typical of endometriosis which may stem from altered progesterone receptor expression8.\n\nFor example, expression of progesterone receptors PR-A and PR-B is altered in endometriotic lesion stromal cells. More specifically, compared with eutopic endometrium, PR-A is markedly reduced and PR-B is absent9. Not only does reduced expression of receptors for this steroid dampen the ability to suppress cytokine production, but reduced progesterone action contributes to elevated local estrogen levels which further drive the endometriotic lesion phenotype and elevated cytokine levels. With this in mind, there has been considerable investigation over the past 20 years examining the roles of specific immune/inflammatory mediators and the potential to target these molecules as novel, estrogen-sparing treatments for this disease. Unfortunately, despite this vast effort, there is still a general sense of uncertainty on which immune/inflammatory mediators appear to be key players in the pathophysiology of endometriosis and the efficacy of targeting these molecules as endometriosis treatment options.\n\n\nTumor necrosis factor and endometriosis\n\nTNF-α, a pro-inflammatory cytokine, was one of the early non-hormonal targets for potential endometriosis therapy10–13. It was first demonstrated to be elevated in the peritoneal fluid14–17 and serum16,17 of women with endometriosis but is now known to be produced by several cell types18, including cells of the endometriotic lesions19. In vitro studies demonstrated that this cytokine stimulated cellular events conducive to the establishment and progression of endometriosis, such as adhesion and induction of protease and inflammatory mediators20–22.\n\nBased upon these observations, initial studies evaluated the efficacy of targeting TNF-α as a potential treatment for endometriosis. The first studies tested a recombinant human TNF-α-binding protein (rhTBP-1)10 in a rat model of endometriosis; these were followed by a series of studies using rhTBP-1 in a baboon model of endometriosis11–13. Unfortunately, studies showing a reduction in disease burden in experimental models have not paralleled studies on efficacy of anti-TNF-α therapy for endometriosis symptomology, as summarized by Lu and colleagues in a recent Cochrane Database review23. Thus, studies on the use of anti-TNF-α have stalled and no new data have emerged to support the use of such compounds for the treatment of symptomatic endometriosis.\n\nThe discrepancy between the encouraging results reported in experimental animal model studies and the lack of an effect detected in clinical trials likely stems from the differences in endpoint analysis. Experimental animal model studies focused primarily on reduced disease burden/lesion size, whereas the clinical trials have focused on the alleviation of pain. Unfortunately, it is unclear whether anti-TNF-α therapy reduced disease burden (stage of endometriosis) in women who received these compounds. We do know from these trials that anti-TNF-α therapy does not reduce pain, which is a chief complaint associated with the disease. The fact that pain is a symptom that is strongly associated with disease presence, but not with disease burden, does not allow conclusions to be drawn with respect to potential impact (or lack of impact) on disease stage in these patients. In animal models, although we do know there is a reduction in disease burden, we do not know whether there is a reduction in pain in those animals treated with anti-TNF-α therapies. Induction of experimental endometriosis in animal models has been demonstrated to elicit pain, initially described in rats by independent groups24,25 and more recently in a mouse model26. Unfortunately, these early studies on anti-TNF-α therapy were conducted prior to the validation of rodent models of pain assessment in animals with experimentally induced endometriosis. One lesson from these studies is that a focus on multiple clinically relevant endpoints in the animal models would be of benefit. Another lesson is that we lack non-surgical clinical biomarkers of disease burden that would be of great use in human studies.\n\nDespite this uncertainty on the role and potential therapeutic benefits of targeting inflammatory mediators such as TNF-α, there is still considerable interest in studying the role of pro-inflammatory mediators in the pathogenesis of endometriosis and the potential benefit of targeting these molecules. Although the initial excitement of anti-TNF-α therapy has waned, additional research on other mediators of inflammation has intensified. Targets getting increased attention are MIF and PGE2.\n\n\nMacrophage migration inhibitory factor and endometriosis\n\nLike TNF-α, MIF is elevated in the peritoneal fluid27, circulation28, and peritoneal macrophages from women with endometriosis29. MIF is also expressed in active and early/stage I endometriotic lesions30, as well as overexpressed in eutopic endometrium in women with the disease31. Within endometriotic lesion cells, MIF is induced by estrogen32, and we have recently demonstrated that MIF expression is associated with endometriotic lesion survival status in women with the disease33.\n\nMIF was originally identified as a potent mitogenic factor for human endothelial cells in vitro and tumor angiogenesis in vivo34. Yang and colleagues demonstrated that, in patients with endometriosis, MIF could stimulate endothelial cell proliferation35. Further supporting a role of MIF in endometriotic lesion survival, MIF has been shown to stimulate PGE2, COX-236, vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), and monocyte chemotactic protein-1 (MCP-1) expression37, as well as the induction of aromatase expression in a feed-forward mechanism32. Interestingly, MIF also stimulates TNF-α secretion38, whereas TNF-α is also capable of inducing MIF production39 in endometrial cells. Thus, it is tempting to speculate that a feed-forward amplification of these cytokines and their downstream pathways exists in endometriosis. Also of relevance to the pathophysiology of endometriosis is the demonstration that many of these MIF-induced factors are associated with a proliferative and angiogenic phenotype conducive to endometriotic establishment or growth (or both)37. As such, there is ample evidence to suggest a strong association between elevated MIF expression/levels and endometriosis in vivo, as well as in vitro evidence which indicates that MIF can induce factors which are believed to be essential for endometriosis development and survival.\n\nBuilding upon these initial observations, several studies have evaluated the efficacy of targeting MIF as a potential endometriosis treatment. In 2011, we first reported the utility of targeting MIF as a potential therapy for endometriosis40. In that study, we used an experimental mouse model of endometriosis in which the females were immune-competent and reproductively intact (non-ovariectomized and non-estrogen-supplemented) and harbored endometriotic lesions derived from donor wild-type mice. We demonstrated that the MIF antagonist, ISO-1, could induce a significant reduction in lesion size. Of potential clinical significance was the finding that ISO-1 reduced lesion burden without affecting reproductive cyclicity or presumed estrogen action40. Using a mouse model for endometriosis in which immune-compromised mice harbored endometriotic lesions derived from human tissue, Khoufache and colleagues41 demonstrated a similar ability of ISO-1 to decrease the number, size, and dissemination of endometriotic lesions. Furthermore, they demonstrated that inhibition of MIF by ISO-1 impedes lesion dynamics by inhibiting cell adhesion, tissue remodeling, angiogenesis, and inflammation, in addition to altering the balance of pro- and anti-apoptotic factors41. More recently, this group provided additional proof of principal by using an ovariectomized, estrogen-supplemented mouse model for endometriosis incorporating Mif-deficient mice as both tissue recipient and tissue donors42. Consistent with previous studies in mouse models40,41, both pharmacologic inhibition of MIF (with ISO-1) and genetic ablation of Mif (Mif-deficient mice) induced a reduction in lesion burden. Of notable interest was the demonstration that Mif-deficient hosts that harbored either normal (expressing Mif) or Mif-deficient lesions had impaired lesion growth, strongly suggesting the critical importance of Mif in the pathogenesis of endometriosis.\n\nInitial studies evaluating ISO-1 as a therapeutic agent for endometriosis treatment are encouraging as the MIF antagonist reduces lesion burden in mouse models which harbor both mouse and human tissue, demonstrating efficacy. Furthermore, this inhibitory effect of MIF antagonism occurs independently of reproductive cyclicity/estrogen levels and action, and may permit continuation of reproductive cycles while relieving disease burden. Clearly, studies are warranted to evaluate whether these beneficial effects of ISO-1 can be extended to alleviating the pain associated with endometriosis in animal models with the extension of MIF antagonist into clinical trials.\n\n\nProstaglandin E2 and endometriosis\n\nIn addition to regulating cytokine production, MIF has been shown to stimulate PGE2 production43. PGE2 has been proposed as a master regulator of endometriosis44 on the basis of its pro-inflammatory actions. PGE2 and the biosynthesis enzymes responsible for its liberation are elevated in human endometriotic lesion tissue45,46 as well as peritoneal macrophages47 and peritoneal fluid48 from women with endometriosis. In vitro studies support a role for PGE2 in the mechanisms conducive to endometriosis establishment and survival. For example, selective inhibition of the PGE2 receptors, prostanoid receptor-2 and (EP2) and EP4, inhibits cellular adhesion, invasion, growth, and survival of human endometriotic epithelial and stromal cells in vitro49–52.\n\nInhibition of PGE2 action has also been associated with favorable outcome in experimental animal models of endometriosis26,53. Using a hamster model of endometriosis, Laschke and colleagues53 demonstrated that administration of the selective COX-2 inhibitor, NS398, induced a marked regression of ectopic lesions by inhibiting angiogenesis and suppressing cellular proliferation and inducing apoptosis. More recently, Arosh and colleagues26 incorporated mouse models of endometriosis and demonstrated that selective inhibition of the PGE2 receptors EP2/EP4 decreased growth and survival, as well as angiogenesis and innervation of ectopic lesions. Furthermore, inhibition of PGE2 signaling was associated with suppression of the pro-inflammatory state of dorsal root ganglia neurons and decreased pelvic pain as well as a decrease in the pro-inflammatory, estrogen-dominant, and progesterone-resistant molecular environment of the eutopic endometrium and ectopic lesions. There are also clinical data which demonstrate that use of rofecoxib, a COX-2 inhibitor (at 25 mg per day for 6 months), resulted in a significant improvement in pelvic pain and dyspareunia after the course of treatment in women with disease, both by comparison with pre- and post-treatment as well as compared with pain assessment in subjects receiving placebo only54. Given that PGE2 is induced by both MIF and TNF-α, it is tempting to speculate that inhibition of these cytokines and the reduction in lesion burden may have been due at least in part to reduction in PGE2 levels or action (or both).\n\n\nEstrogen receptor-beta and endometriosis\n\nAs mentioned earlier in this review, it is well established that endometriosis is an estrogen-dependent disease and that there is a strong connection between estrogen and the inflammatory environment associated with the disease. However, the complex downstream mediators which impart the pathophysiology of the disease are only partially understood. ER-β is one of the two nuclear receptors that mediate estrogen action. Within the context of endometriosis, ER-β is significantly higher (over 100-fold) in endometriotic lesion tissue compared with eutopic endometrium55–57, and this may be due to altered methylation in the gene promoter57. This overexpression of ER-β leads to a decrease in ER-α expression58, resulting in an abnormally high ER-β-to-ER-α ratio which is associated with elevated endometriotic lesion COX-2 levels59. Activation of ER-β has also been demonstrated to induce MIF32 expression by endometriotic lesion cells. Thus, estrogen acting through ER-β-stimulated pathways may play a role in the pathophysiology of endometriosis. Given that endometriosis is an estrogen-dependent disease, inhibition of this pathway might be anticipated to suppress lesion survival and symptoms of endometriosis. If so, one would anticipate that inhibition of ER-β-mediated signaling, though effective in reducing endometriotic lesion burden, may also be associated with an induction of a hypo-estrogenic state, with resultant adverse effects, including menopausal signs and symptoms and loss of reproductive cyclicity.\n\nThree studies to date have evaluated the use of ER-β ligands in the potential treatment of endometriosis using experimental animal models. An early study by Harris and colleagues60 used an ER-β-specific agonist (ERB-041) in an experimental mouse model of endometriosis and reported a regression of ectopic lesion growth. Assessment of lesion tissue (derived from human endometrium) revealed a lack of ER-β expression, leading the authors to conclude that ERB-041 exerted its effects on the host immune system, rather than on the implanted tissue, possibly by induction of apoptosis. Unfortunately, the investigators did not elaborate on the mechanism by which this occurred. As activation of ER-β decreases ER-α expression58, it may be possible that downregulation of ER-α contributed to these observations.\n\nMore recently, Zhao and colleagues61 elegantly dissected the role of both ER-α and ER-β signaling by using an experimental mouse model of endometriosis incorporating the novel ER ligands chloroindazole (CLI) (exhibits ER-β-dependent activity) and oxabicycloheptene sulfonate (OBHS) (greater ER-α-preferential binding selectivity) which exhibit both anti-estrogenic and anti-inflammatory activity. Most importantly, both CLI and OBHS induced lesion regression and suppression of inflammatory events associated with endometriosis without disrupting normal reproductive cyclicity and fertility. Thus, the anti-estrogenic/antagonistic effect of these ligands suggests that the ER-β (and ER-α) pathway is involved in the pathogenesis of endometriosis, and that the effects of estrogen antagonism can be separated between those that impact inflammation and lesion regression and those that regulate reproductive cyclicity and fertility.\n\nThis postulate is further supported by the work of Han and colleagues62, who demonstrated that activation of the ER-β pathway may contribute to the pathogenesis of endometriosis. Using experimental mouse models of endometriosis which incorporate genetically modified mice in which ER-α and ER-β are conditionally deleted, these investigators demonstrated that use of the ER-β antagonist, PHTPP, was associated with a regression of ectopic lesions. These investigators went on to dissect the mechanism by using experimental endometriosis models that incorporated genetically modified mice which either overexpressed ER-β, or had ER-α or ER-β (or both) deleted from uterine/endometriotic tissue. This study demonstrated that ER-β is responsible for inhibiting endometriotic cell apoptosis and increases cytokine production to enhance cellular adhesion and proliferation as well as enhance epithelial-mesenchymal transition signaling to increase cell invasion. As suggested by Han and colleagues, and supported by the study by Zhoa and colleagues61, targeting ER-β may have beneficial effects on lesion growth/survival as well as the potential to improve infertility at the level of the eutopic endometrium62, or at least spare reproductive competency while reducing lesion burden. The potential efficacy of targeting ER-β/ER-α with these novel ligands to reduce pain associated with endometriosis remains to be determined, as does the assessment of the potential impact on bone density.\n\n\nSummary\n\nFor over two decades, the role of inflammatory mediators and the potential to target them as a non-hormonal means of treating endometriosis have been explored. Early studies focusing on TNF-α appeared promising on the basis of effects on lesions in experimental animal models but failed to produce clinical results on pain symptoms. More recent focus has turned toward MIF and PGE2 as potential targets for endometriosis treatment. Much like earlier studies focusing on TNF-α, experimental model studies have yielded promising results on their ability not only to suppress lesion growth but also to reduce pelvic pain, both independently of reproductive cyclicity. As yet, confirmatory studies in human subjects remain to be initiated. In addition to MIF and PGE2, the ER-β pathway has emerged as a potential target for endometriosis treatment. Of interest is the finding that the ER-β pathway appears to mediate many of the cytokines described in this review in modulating endometriotic lesion growth in animal models (summarized in Figure 1).\n\n(a) Estrogen stimulates the establishment, growth, and survival of endometriotic tissue through the induction of tumor necrosis factor-alpha (TNF-α), macrophage migration inhibitory factor (MIF), and prostaglandin E2 (PGE2) (black arrows) as well as through other estrogen receptor-alpha (ER-α)- and ER-β-dependent pathways (orange and pink arrows, respectively). (b) Inhibition of estrogen, TNF-α, MIF, and PGE2 leads to reduced endometriosis burden in experimental animal models of endometriosis. Broken lines indicate inhibition of endometriotic lesion burden by antagonism of estrogen, TNF-α, MIF and/or PGE2 signaling. “- Pain” indicates those specified antagonists which were demonstrated to reduce lesion burden and pain in experimental animal models of endometriosis. “+ Reproductive cyclicity” indicates those specified antagonists which reduced endometriosis burden but did not negatively impact reproductive cyclicity/fertility in experimental animal models of endometriosis.\n\nWhat we have learned in recent years is that estrogen action within the pathogenesis of endometriosis can be partitioned into inflammatory pathways that drive lesion survival and those steroid hormone pathways that modulate reproductive cyclicity. With recent advances in our ability to dissect the estrogen-regulated pathways by using novel pharmacologic and genetic tools, we have learned that the most effective estrogen-sparing target for endometriosis treatment may be an estrogen receptor itself.\n\n\nAbbreviations\n\nCLI, chloroindazole; COX-2, cyclooxygenase-2; EP2, prostanoid receptor-2; EP4, prostanoid receptor-4; ER-α, estrogen receptor-alpha; ER-β, estrogen receptor-beta; ISO-1, (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester; MIF, macrophage migration inhibitory factor (human); Mif, macrophage migration inhibitory factor (murine); OBHS, oxabicycloheptene sulfonate; PGE2, prostaglandin E2; PHTPP, 4-[2-Phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol; rhTMP-1, recombinant human tumor necrosis factor-alpha-binding protein; TNF-α, tumor necrosis factor-alpha.", "appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThe work cited in this review was supported in part by the Eunice Kennedy Shriver National Institute of Child Health and Human Development grants HD069043 and HD073733 to Warren B. 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PubMed Abstract \| Publisher Full Text\n\nNothnick WB, Colvin A, Cheng KF, et al.: Inhibition of macrophage migration inhibitory factor reduces endometriotic implant size in mice with experimentally induced disease. J Endometr. 2011; 3(3): 135–142. PubMed Abstract \| Free Full Text\n\nKhoufache K, Bazin S, Girard K, et al.: Macrophage migration inhibitory factor antagonist blocks the development of endometriosis in vivo. PLoS One. 2012; 7(5): e37264. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nRakhila H, Girard K, Leboeuf M, et al.: Macrophage migration inhibitory factor is involved in ectopic endometrial tissue growth and peritoneal-endometrial tissue interaction in vivo: a plausible link to endometriosis development. PLoS One. 2014; 9(10): e110434. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nLeng L, Metz CN, Fang Y, et al.: MIF signal transduction initiated by binding to CD74. J Exp Med. 2003; 197(11): 1467–1476. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWu MH, Lu CW, Chuang PC, et al.: Prostaglandin E2: the master of endometriosis? Exp Biol Med (Maywood). 2010; 235(6): 668–677. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nOta H, Igarashi S, Sasaki M, et al.: Distribution of cyclooxygenase-2 in eutopic and ectopic endometrium in endometriosis and adenomyosis. Hum Reprod. 2001; 16(3): 561–566. PubMed Abstract \| Publisher Full Text\n\nRakhila H, Carli C, Daris M, et al.: Identification of multiple and distinct defects in prostaglandin biosynthetic pathways in eutopic and ectopic endometrium of women with endometriosis. Fertil Steril. 2013; 100(6): 1650–9.e1–2. PubMed Abstract \| Publisher Full Text\n\nWu MH, Sun HS, Lin CC, et al.: Distinct mechanisms regulate cyclooxygenase-1 and -2 in peritoneal macrophages of women with and without endometriosis. Mol Hum Reprod. 2002; 8(12): 1103–1110. PubMed Abstract \| Publisher Full Text\n\nDe Leon FD, Vijayakumar R, Brown M, et al.: Peritoneal fluid volume, estrogen, progesterone, prostaglandin, and epidermal growth factor concentrations in patients with and without endometriosis. Obstet Gynecol. 1986; 68(2): 189–194. PubMed Abstract\n\nBanu SK, Lee J, Speights VO Jr, et al.: Selective inhibition of prostaglandin E2 receptors EP2 and EP4 induces apoptosis of human endometriotic cells through suppression of ERK1/2, AKT, NFkappaB, and beta-catenin pathways and activation of intrinsic apoptotic mechanisms. Mol Endocrinol. 2009; 23(8): 1291–1305. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nLee J, Banu SK, Burghardt RC, et al.: Selective inhibition of prostaglandin E2 receptors EP2 and EP4 inhibits adhesion of human endometriotic epithelial and stromal cells through suppression of integrin-mediated mechanisms. Biol Reprod. 2013; 88(3): 77. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nLee J, Banu SK, Rodriguez R, et al.: Selective blockade of prostaglandin E2 receptors EP2 and EP4 signaling inhibits proliferation of human endometriotic epithelial cells and stromal cells through distinct cell cycle arrest. Fertil Steril. 2010; 93(8): 2498–2506. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nLee J, Banu SK, Subbarao T, et al.: Selective inhibition of prostaglandin E2 receptors EP2 and EP4 inhibits invasion of human immortalized endometriotic epithelial and stromal cells through suppression of metalloproteinases. Mol Cell Endocrinol. 2011; 332(1–2): 306–313. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nLaschke MW, Elitzsch A, Scheuer C, et al.: Selective cyclo-oxygenase-2 inhibition induces regression of autologous endometrial grafts by down-regulation of vascular endothelial growth factor-mediated angiogenesis and stimulation of caspase-3-dependent apoptosis. Fertil Steril. 2007; 87(1): 163–171. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nCobellis L, Razzi S, De Simone S, et al.: The treatment with a COX-2 specific inhibitor is effective in the management of pain related to endometriosis. Eur J Obstet Gynecol Reprod Biol. 2004; 116(1): 100–102. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nBrandenberger AW, Lebovic DI, Tee MK, et al.: Oestrogen receptor (ER)-alpha and ER-beta isoforms in normal endometrial and endometriosis-derived stromal cells. Mol Hum Reprod. 1999; 5(7): 651–655. PubMed Abstract \| Publisher Full Text\n\nFujimoto J, Hirose R, Sakaguchi H, et al.: Expression of oestrogen receptor-alpha and -beta in ovarian endometriomata. Mol Hum Reprod. 1999; 5(8): 742–747. PubMed Abstract \| Publisher Full Text\n\nXue Q, Lin Z, Cheng YH, et al.: Promoter methylation regulates estrogen receptor 2 in human endometrium and endometriosis. Biol Reprod. 2007; 77(4): 681–687. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nTrukhacheva E, Lin Z, Reierstad S, et al.: Estrogen receptor (ER) beta regulates ERalpha expression in stromal cells derived from ovarian endometriosis. J Clin Endocrinol Metab. 2009; 94(2): 615–622. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nBulun SE, Monsavais D, Pavone ME, et al.: Role of estrogen receptor-β in endometriosis. Semin Reprod Med. 2012; 30(1): 39–45. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHarris HA, Bruner-Tran KL, Zhang X, et al.: A selective estrogen receptor-beta agonist causes lesion regression in an experimentally induced model of endometriosis. Hum Reprod. 2005; 20(4): 936–941. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nZhao Y, Gong P, Chen Y, et al.: Dual suppression of estrogenic and inflammatory activities for targeting of endometriosis. Sci Transl Med. 2015; 7(271): 271ra9. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nHan SJ, Jung SY, Wu SP, et al.: Estrogen Receptor β Modulates Apoptosis Complexes and the Inflammasome to Drive the Pathogenesis of Endometriosis. Cell. 2015; 163(4): 960–974. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation" }	[ { "id": "12517", "date": "18 Feb 2016", "name": "Steven L Young", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12518", "date": "18 Feb 2016", "name": "Bruce A Lessey", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12519", "date": "18 Feb 2016", "name": "Kaylon L Bruner-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", "responses": [] }, { "id": "12520", "date": "18 Feb 2016", "name": "Joe A. Arosh", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-186
https://f1000research.com/articles/5-185/v1	17 Feb 16	{ "type": "Review", "title": "Plant-based biofuels", "authors": [ "Elizabeth E. Hood" ], "abstract": "This review is a short synopsis of some of the latest breakthroughs in the areas of lignocellulosic conversion to fuels and utilization of oils for biodiesel. Although four lignocellulosic ethanol factories have opened in the USA and hundreds of biodiesel installations are active worldwide, technological improvements are being discovered that will rapidly evolve the biofuels industry into a new paradigm. These discoveries involve the feedstocks as well as the technologies to process them.", "keywords": [ "Plant-based biofuels", "lignocellulosic conversion", "biodiesel", "biofuels" ], "content": "Introduction\n\nAn important mitigation strategy for the impact of fossil fuels on the environment is to use biofuels from renewable sources for transportation. Biofuels from plants represent the most abundant source of renewable fuels, offering the manufacture of ethanol and butanol (as gasoline additives) and long-chain hydrocarbons (for diesel additives or as jet fuels) from starch, cellulose, hemicellulose, and oils. The source of the energy captured by plants is the sun, which will be a constant source of energy for the next few billion years. The carbon released from the burning of biofuels is continually cycled rather than being released from ancient fixed carbon sources, as is the case for fossil petroleum and natural gas. The problem is that the cost of production of fuels from lignocellulose and plant oils is high and this nascent industry cannot compete with oil prices.\n\nCurrent progress: For the past two decades, ethanol has been produced primarily from cornstarch and cane sugar. Fourteen billion gallons of ethanol were produced in the USA from cornstarch in 2014 (Figure 1). Also shown in Figure 1 is that corn-based ethanol production has plateaued (http://www.ethanolrfa.org/wp-content/uploads/2015/09/23d732bf7dea55d299_3wm6b6wwl.pdf). Approximately 40% of the current USA corn crop is used to produce ethanol and is not likely to expand anymore, because the remainder of the crop is being used for animal feed and human food. Ethanol is produced from cane sugar in Brazil at a level of 7.3 billion gallons in 2014 (http://sugarcane.org/sugarcane-products/ethanol). Together, Brazil and the USA produce more than 90% of the world’s supply of ethanol.\n\nProduction has increased from approximately 4 billion gallons (15.1 billion liters) in 2005 to over 14 billion gallons (53 billion liters) in 2014.\n\nBiodiesel is a renewable fuel that has received considerable attention recently because it is also non-polluting. It is carbon neutral because the carbon present in vehicle exhaust was recently fixed from atmospheric carbon1. Biodiesel can be manufactured from numerous oils and fats including virgin vegetable oils, such as canola, soybean, and camelina, from waste cooking oils, or from animal fats, such as tallow or lard. The global biodiesel industry has grown considerably over the last several years2, although since 2008 a dip occurred based on world economic recession. Europe has produced the greatest volume of biodiesel over the years, followed by the USA. Worldwide production in 2012 comprised 6 billion gallons (22.5 billion liters) (http://www.uabio.org/img/files/docs/140526-wba-gbs-2014.pdf). Production in the USA in 2012 was approximately 0.89 billion gallons but increased to over 1.25 billion gallons in 2014 (Energy Information Administration as shown in Figure 2).\n\nProduction has increased from 112 million gallons in 2005 to over 1.3 billion gallons in 2014. A large dip in production was seen from 2008 to 2010 during the economic recession.\n\nThe Renewable Fuel Standard II (RFS II): RFS II is the motivation for increasing the production of renewable fuels from green plants (http://www.ethanolrfa.org/policy/regulations/renewable-fuel-standard/). This standard was set in 2005 and revised in 2007 to mandate quantities of renewable fuels to be incorporated into the transportation industry in the USA. The goal for 2022 is set at 36 billion gallons of renewable fuels, with 16 billion gallons required to be from lignocellulosic feedstocks, and 1 billion gallons per year of biodiesel. Additionally, 58% of the fuels produced by 2022 should be “advanced biofuels”, e.g. non-starch ethanol or other types of fuels such as long-chain hydrocarbons or butanol that achieve a 50% reduction in greenhouse gas emissions. The feedstocks for these fuels are lignocellulose and oils. However, intense research is necessary to make this cost-competitive. Breakthroughs are being made in feedstock structure, ease of processing, efficiency of conversion, co-product manufacture, and sustainability. As these discoveries come together, they can be incorporated into new industrial applications.\n\n\nLignocellulosic biofuels\n\nResearch Roadmap: In 2007, the US Department of Energy conducted a workshop to assess the major roadblocks to the production of lignocellulosic biofuels (https://www1.eere.energy.gov/bioenergy/pdfs/obp_roadmapv2_web.pdf). The purpose of the workshop was to guide research and development activities that would enable the biofuels industry in an accelerated time frame. Barriers were identified for feedstocks, deconstruction and conversion, infrastructure, and sustainability of the industry. In the case of feedstocks, yield of biomass, broader feedstock variety base, reduction of recalcitrance, and improved nutrient and water use efficiency were targets (Figure 3). For deconstruction and conversion to biofuels, reducing the cost of enzymes and improving conversion efficiencies were targets to reduce cost, as well as broadening the number and condition of crops that could be used as feedstock. An overlying goal is to improve the sustainability of the industry, from crop growth and harvest through to product manufacture and feedstock utilization. Although infrastructure and outreach were part of the roadmap, they will not be addressed in this review. This Roadmap was used to establish a call for proposals to fund three Bioenergy Research Centers, resulting in the Great Lakes Bioenergy Research Center, the BioEnergy Science Center, and the Joint BioEnergy Institute. These three centers along with numerous other smaller entities are making significant progress toward addressing these barriers through targeted research programs.\n\nA variety of feedstocks can be harvested, ground, and fed into unit operations that allow the tight cell wall structures to be loosened (pretreatment) for enzymatic deconstruction, or for chemical deconstruction to be accomplished. Resulting chemicals comprise five- and six-carbon sugars and lignin in a range of polymerization degrees (monomers to long polymers). Biofuels and biobased products are manufactured from these raw materials through a variety of microbial or chemical conversion processes. Research in each of these areas is highlighted in the upper boxes and discussed in the review.\n\nFeedstock recalcitrance and productivity: The first four lignocellulosic biofuel manufacturing plants have been opened in the USA. Their biomass feedstocks comprise residual materials from food crops and forestry operations. Two installations in Iowa owned by Poet, LLC (Sioux Falls, SD) and DuPont (Wilmington, DE) use corn stover and residual materials as their feedstock. The Abengoa Bioenergy Biomass of Kansas (ABBK) installation in Hugoton, KS, used primarily wheat straw (the plant is now closed and is for sale, at time of writing), although other feedstocks were used as available. INEOS Bio in Vero Beach, FL, utilizes vegetative and wood waste. The next generation of biofuel installations likely will use dedicated biomass crops such as poplar, switchgrass, sorghum, and miscanthus, because they are in intense research programs to improve their yield and digestibility.\n\nLignin is the primary molecule that contributes to recalcitrance (lack of digestibility) because of its complex structure and prevalent ether bonds that are difficult to break. It is also cross-linked to many potentially digestible cell wall polymers3. A recent breakthrough in lowering recalcitrance is to engineer plants to have different linkages in lignin4–7. The enzyme monolignol ferulate transferase introduces chemically labile linkages into the lignin backbone, facilitating the release of five- and six-carbon sugars from cell wall polysaccharides after mild pretreatment6. This Zip Lignin™ (https://www.glbrc.org/research/technologies/grass-modified-easier-bioprocessing) has multiple ester linkages that are far easier to break than the more common ether linkages6. Down-regulation of caffeic acid O-methyltransferase (COMT) in switchgrass also lowers lignin8. Although only a modest amount lower, these plants have a reduced syringyl:guaiacyl lignin monomer ratio and increased ethanol yield by up to 38% using conventional biomass fermentation processes8.\n\nSeveral other cell wall mutations or manipulations have increased productivity of biomass and/or lowered biomass recalcitrance. Enhancing syringyl lignin in pine tracheary elements could enhance bioprocessing9, enhanced content of sinapaldehyde in lignin in Arabidopsis leads to enhanced digestibility of cell walls10, and adding bacterial genes into Arabidopsis can form oxidized lignin that is more easily digested11. Lowering total lignin content rather than altering monomer ratios through manipulations of the p-coumaroyl quinate/shikimate 3′-hydroxylase and cinnamate 4-hydroxylase genes in Eucalyptus significantly lowered recalcitrance12. Baxter and colleagues found that over-expression of the switchgrass transcription factor PvMYB4, which acts as a transcriptional repressor of many lignin biosynthetic genes, reduced lignin in transgenic switchgrass plants by as much as 50%13. While some of the transgenic plants were less field-hardy, one robust transgenic line had 63% greater biomass and yielded 32% more biofuel.\n\nIn efforts to understand pectin synthesis and accumulation, galacturonosyl transferase (GAUT) gene family members were over- and under-expressed in switchgrass. Under-expression through RNA interference (RNAi) of GAUT12.1 lowered pectin content and surprisingly increased glucose release by as much as 8% over controls without compromising growth, actually increasing plant height and stem diameter14,15. These knockdown mutants had less xylan as well as less pectin, although total lignin content was the same as in controls.\n\nIn addition to manipulating cell wall recalcitrance, plants have been manipulated to exhibit traits that increase biomass production. For example, Brachypodium distachyon plants in which phytochrome C is down-regulated have greatly delayed flowering, contributing to enhanced biomass accumulation16. Quantitative trait loci (QTLs) were identified in switchgrass that define biomass yield and plant height17. QTLs are groups of genes that co-segregate and act together to generate the phenotype of interest.\n\nDeconstruction and fuel production: A number of different pretreatment regimes have been explored in detail to be used with various enzyme systems. Ammonia fiber expansion (90–120°C; 250–400 psi) is highly effective on grasses such as corn stover and switchgrass but does not produce a lignin fraction that can be used for co-product manufacture18. Lignin is desirable because its monomers can be used as a raw material for many more complex chemicals. Because the ammonia can be quantitatively recovered, it is relatively cost effective. Alkaline hydrogen peroxide is used in pulp-bleaching and is an effective delignification agent19. However, the high concentration of hydrogen peroxide made this method cost-prohibitive. The addition of a small amount of copper ions significantly improves the lignin extraction, thus making it highly effective for woody biomass pretreatment20. These treatments usually are followed by treatment with enzyme mixtures to deconstruct the cellulose and hemicellulose from the cell walls, producing sugars for fermentation, though in order for the enzymes to function, the biomass alkalinity must be neutralized, adding further cost to the processing and generating additional waste.\n\nA promising new solvent for treating biomass is γ-valerolactone, or GVL21. It is derived from the biomass itself and appears to pretreat any type of biomass22 yielding sugar, lignin, and mineral salt streams that can be separated from the reaction mix23. Ionic liquids (ILs) offer a pretreatment strategy that has many advantages, including significant enhancement in the rate of enzyme hydrolysis of the cellulose component of switchgrass and a 96% recovery of glucan in 24 hours24. However, IL is quite expensive at its current state of development.\n\nPretreated biomass is generally deconstructed with enzymes, whether these are mixtures isolated from fungal cultures, multifunctional enzymes isolated from microorganisms, or mixtures isolated from live bioprocessing organisms growing on the biomass. To use enzymes cost-effectively for biomass conversion prior to fermentation, it is estimated that the cost of the enzymes should be approximately $0.10 per gallon of ethanol25,26. For the past 15 years, intense research on enzyme production platforms has yielded fungal enzyme mixtures that do not meet these cost requirements and in fact also require a huge infrastructure for production. A relatively new technology utilizes genetically engineered plant seeds (primarily maize) to accumulate industrial enzymes25. The plant seed production system is more cost-competitive but has not been tested at scale for efficacy25. Current research efforts are in multifunctional enzymes (USP application # 2014/0079683) and combined bioprocessing organisms, the latter of which can decompose plant polymers as well as ferment them into biofuels27.\n\nOnce the biomass has been deconstructed into sugars, those sugars should be fermented into fuels such as ethanol, butanol, or longer-chain hydrocarbons. These products are currently manufactured through bacterial (Clostridium sp.) or yeast fermentation. However, new microorganisms, many of which are thermophilic bacteria, are under investigation as potential biofuel-producing microbes27. Significant changes in their metabolic networks are required to allow them to produce a single product, such as ethanol, at high titer and without inhibition by the product. Additional inhibition is seen as a result of pretreatment of various lignocellulosic feedstocks28. These thermophiles, such as Caldicellulosiruptor bescii, can also be used to probe plant cell wall structure through mutated enzyme activities29.\n\nSustainability: Production of biomass for non-food uses, such as for biofuels and biobased products, has faced a fierce debate with advocates for food production on arable agricultural land, as well as land use changes. Thus, research on increasing the productivity of biomass crops, use of marginal lands for production, fewer inputs such as water and nutrients, and recovering maximal sugars and co-products are key outcomes for achieving the goal of production of billions of gallons of biofuels from lignocellulosic feedstock. Life cycle assessments of various biomass crops, produced in different soils under different environmental conditions, are being completed to understand the best cropping systems with the best environmental outcome30. In addition, the square footage of the biorefinery that produces the fuel can have a major impact on sustainability, impacting the transportation radius for bringing biomass from the surrounding area31. Growth and nutrient-use advantages are being seen through inoculation with endophytic fungi32, and manipulation of transcription factor genes that are important for nutrient cycling during senescence may improve nutrient-use efficiency in perennial plants such as switchgrass33.\n\n\nPlant oils to biofuels\n\nBiodiesel-esters from triacylglycerol (TAG) and diacylglycerol (DAG, Figure 4): Biodiesel is predominantly commercially manufactured through treatment of feedstock oil or fat with alcohols and chemical catalysts such as sodium or potassium hydroxide. Although this method can be commercially profitable, this is true only when certain conditions are met1,34,35. First, virgin plant oils are generally too expensive for the process and the more cost-effective feedstock is usually recycled cooking oil or other used oils2. Second, the feedstock oil must be low in free fatty acids (FFAs, less than 2%), or those FFAs must be removed prior to chemical reactions. If not removed, they saponify (i.e. make soaps), forming emulsions with the catalyst, and make the biodiesel more difficult to separate from the contaminants36. Third, phospholipids must also be removed because they result in foaming or emulsions, which are difficult to remove from the finished biodiesel. Additional considerations are that the process is energy intensive and the alkaline waste water requires treatment for reuse2. The primary products of fats and oils are fatty acid methyl esters (FAMEs, biodiesel, Figure 4)35, which must be separated from the contaminating glycerol, water, and acid or base catalyst.\n\nTop: TAG (triacylglycerol); DAG (diacylglycerol); center: phospholipid; bottom: fatty acid methyl esters (FAMEs) or biodiesel produced through trans-esterification. Phospholipase yields DAG and R-phosphate. Lipase yields FAMEs.\n\nUsing enzymes rather than non-enzymatic catalysts can address several of these issues. Biological processes in any type of industrial reaction can promote more specific products from the feedstock and can avoid co-product formation2. Additionally, the products are regiospecific, i.e. the enzyme selectively generates one constitutional isomer rather than the other. Moreover, they represent an environmentally friendly process for making biodiesel that yields methyl esters from FFAs and from the DAG liberated by phospholipases from phospholipids, increasing the overall biodiesel yield from the feedstock oil.\n\nLipases and phospholipases: Lipases (EC 3.1.1.3) are carboxylesterases, the most important group of biocatalysts for biotechnological applications in organic chemistry37. Numerous lipases have been identified that are active on various substrates to a greater or lesser degree. Commercial lipases are currently produced through microbial fermentation and used industrially for the synthesis of biopolymers, biodiesel, agrochemicals, flavor compounds, and enantiopure pharmaceuticals. They catalyze highly stereospecific reactions and have very few side reactions. For biodiesel application, large volumes of low-cost enzymes that can be immobilized onto appropriate substrates will enable enzymatic biodiesel production on a large scale. Phospholipase, in particular phospholipase C (PLC, EC 3.1.4.3), catalyzes the hydrolysis of phosphate-R groups from phospholipids (Figure 4), resulting in DAG. DAG is an excellent substrate for the lipases in trans-esterification reactions, improving the yield of biodiesel from oil feedstocks. The enzyme has been produced commercially in Pichia pastoris (http://www.fao.org/fileadmin/templates/agns/pdf/jecfa/cta/69/Phospholipase_C.pdf).\n\nOil degumming using phospholipase: Degumming is an essential process for purifying waste cooking and vegetable oils to produce a quality feedstock for biodiesel38. The process removes the contaminating phosphorus-containing lipids (Figure 4), which act as emulsifiers and trap neutral oil, resulting in loss of biodiesel feedstock. For chemically catalyzed biodiesel, the phospholipid content of the oil must be less than 10 parts per million (ppm) and is often removed from the feedstock by mixing a small volume of water with the oil. The majority of these aliphatic phospholipids form a gummy mass that can be removed by centrifugation or filtration. Phospholipase A1, A2, or C can also be used to remove phospholipids. The enzyme releases 1,2-DAG that can be processed through either enzymatic or chemical processes to FAMEs.\n\nLipase can produce FAME through esterification and trans-esterification: The primary reaction to produce biodiesel is to convert di- and tri-glycerides (Figure 4) into methyl esters. Biodiesel comprises these FAMEs. Chemical conversion uses a large excess of methanol to push the reaction toward the product and its co-product glycerol36. Using lipase allows for stoichiometric and slow addition of methanol to the reaction mixture because the enzyme itself pushes the reaction toward the regiospecific product.\n\n\nConclusions\n\nPlants as sources of biofuels have many advantages, particularly a neutral carbon balance. Although much rhetoric has surfaced to discourage the growth of biofuel crops because they utilize farmland that should be dedicated to food crops, in reality the productivity and yield of fuels from dedicated energy crops appears to be on a steep, upward trajectory. Moreover, the technology to produce ethanol and biodiesel from plant biomass has progressed at a phenomenal rate, generating confidence that the industry will be profitable and sustainable. Because biomass varies widely in chemical composition and structure, and processes applicable to each biomass type vary as well, smaller biorefineries may be the standard of this industry rather than requiring large biorefineries to reach economic feasibility31,39. This conclusion is based on the cited studies that look at specific processes applied to specific biomass types, such as woody biomass and GVL, which would enable a biorefinery to produce fuels efficiently from a specific biomass type. Although not discussed here, a contributing factor to the profitability of the industry will be the manufacture of co-products from the biomass: carbon fibers, fillers, resins, or polymer blends from lignin22,40,41 or pre-extracted plastics42 or enzymes43 prior to deconstructing the biomass.\n\n\nAbbreviations\n\nDAG, Diacylglycerol; FAME, Fatty Acid Methyl Ester; FFA, Free Fatty Acids; GAUT, Galacturonosyl transferase; GVL, γ-valerolactone; IL, Ionic liquid.", "appendix": "Competing interests\n\n\n\nThe author declares that they have no competing interests.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nI wish to thank the F1000 staff for inviting me to write this review.\n\n\nReferences\n\nRanganathan SV, Narasimhan SL, Muthukumar K: An overview of enzymatic production of biodiesel. Bioresour Technol. 2008; 99(10): 3975–3981. 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Proc Natl Acad Sci U S A. 2015; 112(19): 6218–6223. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nAnderson NA, Tobimatsu Y, Ciesielski PN, et al.: Manipulation of Guaiacyl and Syringyl Monomer Biosynthesis in an Arabidopsis Cinnamyl Alcohol Dehydrogenase Mutant Results in Atypical Lignin Biosynthesis and Modified Cell Wall Structure. Plant Cell. 2015; 27(8): 2195–2209. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nTsuji Y, Vanholme R, Tobimatsu Y, et al.: Introduction of chemically labile substructures into Arabidopsis lignin through the use of LigD, the Cα-dehydrogenase from Sphingobium sp. strain SYK-6. Plant Biotechnol J. 2015; 13(6): 821–832. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nSykes RW, Gjersing EL, Foutz K, et al.: Down-regulation of p-coumaroyl quinate/shikimate 3'-hydroxylase (C3'H) and cinnamate 4-hydroxylase (C4H) genes in the lignin biosynthetic pathway of Eucalyptus urophylla × E. grandis leads to improved sugar release. Biotechnol Biofuels. 2015; 8: 128. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nBaxter HL, Poovaiah CR, Yee KL, et al.: Field Evaluation of Transgenic Switchgrass Plants Overexpressing PvMYB4 for Reduced Biomass Recalcitrance. Bioenerg Res. 2015; 8(3): 910–21. Publisher Full Text \| Faculty Opinions Recommendation\n\nBiswal AK, Hao Z, Pattathil S, et al.: Downregulation of GAUT12 in Populus deltoides by RNA silencing results in reduced recalcitrance, increased growth and reduced xylan and pectin in a woody biofuel feedstock. Biotechnol Biofuels. 2015; 8: 41. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nHao Z, Avci U, Tan L, et al.: Loss of Arabidopsis GAUT12/IRX8 causes anther indehiscence and leads to reduced G lignin associated with altered matrix polysaccharide deposition. Front Plant Sci. 2014; 5: 357. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nWoods DP, Ream TS, Minevich G, et al.: PHYTOCHROME C is an essential light receptor for photoperiodic flowering in the temperate grass, Brachypodium distachyon. Genetics. 2014; 198(1): 397–408. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nSerba DD, Daverdin G, Bouton JH, et al.: Quantitative Trait Loci (QTL) Underlying Biomass Yield and Plant Height in Switchgrass. Bioenerg Res. 2015; 8(1): 307–24. Publisher Full Text \| Faculty Opinions Recommendation\n\nGarlock RJ, Balan V, Dale BE: Optimization of AFEX™ pretreatment conditions and enzyme mixtures to maximize sugar release from upland and lowland switchgrass. Bioresour Technol. 2012; 104: 757–768. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nLi Z, Chen CH, Hegg EL, et al.: Rapid and effective oxidative pretreatment of woody biomass at mild reaction conditions and low oxidant loadings. Biotechnol Biofuels. 2013; 6(1): 119. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nLi Z, Chen CH, Liu T, et al.: Catalysis with CuII(bpy) improves alkaline hydrogen peroxide pretreatment. Biotechnol Bioeng. 2013; 110(4): 1078–1086. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nBond JQ, Alonso DM, Wang D, et al.: Integrated catalytic conversion of gamma-valerolactone to liquid alkenes for transportation fuels. Science. 2010; 327(5969): 1110–1114. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nLuterbacher JS, Alonso DM, Rand JM, et al.: Solvent-enabled nonenyzmatic sugar production from biomass for chemical and biological upgrading. ChemSusChem. 2015; 8(8): 1317–1322. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nHan J, Sen SM, Alonso DM, et al.: A strategy for the simultaneous catalytic conversion of hemicellulose and cellulose from lignocellulosic biomass to liquid transportation fuels. Green Chem. 2014; 16(2): 653–61. Publisher Full Text\n\nLi C, Knierim B, Manisseri C, et al.: Comparison of dilute acid and ionic liquid pretreatment of switchgrass: Biomass recalcitrance, delignification and enzymatic saccharification. Bioresour Technol. 2010; 101(13): 4900–4906. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nHoward JA, Nikolov Z, Hood EE: Enzyme Production Systems for Biomass Conversion. In Plant Biomass Conversion. (eds. E.E. Hood, P. Nelson & R. Powell). Wiley, 2011; 227–253. Publisher Full Text\n\nKlein-Marcuschamer D, Oleskowicz-Popiel P, Simmons BA, et al.: The challenge of enzyme cost in the production of lignocellulosic biofuels. Biotechnol Bioeng. 2012; 109(4): 1083–1087. PubMed Abstract \| Publisher Full Text\n\nOlson DG, Sparling R, Lynd LR: Ethanol production by engineered thermophiles. Curr Opin Biotechnol. 2015; 33: 130–141. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nKeating DH, Zhang Y, Ong IM, et al.: Aromatic inhibitors derived from ammonia-pretreated lignocellulose hinder bacterial ethanologenesis by activating regulatory circuits controlling inhibitor efflux and detoxification. Front Microbiol. 2014; 5: 402. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nYoung J, Chung D, Bomble YJ, et al.: Deletion of Caldicellulosiruptor bescii CelA reveals its crucial role in the deconstruction of lignocellulosic biomass. Biotechnol Biofuels. 2014; 7(1): 142. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nSinistore JC, Reinemann DJ, Izaurralde RC, et al.: Life Cycle Assessment of Switchgrass Cellulosic Ethanol Production in the Wisconsin and Michigan Agricultural Contexts. Bioenerg Res. 2015; 8(3): 897–909. Publisher Full Text\n\nKim S, Dale BE: All biomass is local: The cost, volume produced, and global warming impact of cellulosic biofuels depend strongly on logistics and local conditions. Biofuels Bioprod Bioref. 2015; 9(4): 422–34. Publisher Full Text\n\nRay P, Ishiga T, Decker SR, et al.: A Novel Delivery System for the Root Symbiotic Fungus, Sebacina vermifera, and Consequent Biomass Enhancement of Low Lignin COMT Switchgrass Lines. Bioenerg Res. 2015; 8(3): 922–33. Publisher Full Text \| Faculty Opinions Recommendation\n\nYang J, Worley E, Torres-Jerez I, et al.: PvNAC1 and PvNAC2 Are Associated with Leaf Senescence and Nitrogen Use Efficiency in Switchgrass. Bioenerg Res. 2015; 8(2): 868–80. Publisher Full Text \| Faculty Opinions Recommendation\n\nRibeiro BD, de Castro AM, Coelho MA, et al.: Production and use of lipases in bioenergy: a review from the feedstocks to biodiesel production. Enzyme Res. 2011; 2011: 615803. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSotoft LF, Rong BG, Christensen KV, et al.: Process simulation and economical evaluation of enzymatic biodiesel production plant. Bioresour Technol. 2010; 101(14): 5266–5274. PubMed Abstract \| Publisher Full Text\n\nMa F, Hanna MA: Biodiesel production: a review. Bioresour Technol. 1999; 70(1): 1–15. Publisher Full Text\n\nJaeger KE, Eggert T: Lipases for biotechnology. Curr Opin Biotechnol. 2002; 13(4): 390–397. PubMed Abstract \| Publisher Full Text\n\nWatanabe Y, Shimada Y, Sugihara A, et al.: Conversion of degummed soybean oil to biodiesel fuel with immobilized Candida antarctica lipase. J Mol Catal B Enzym. 2002; 17(3–5): 151–5. Publisher Full Text\n\nKim S, Dale BE: Comparing alternative cellulosic biomass biorefining systems: Centralized versus distributed processing systems. Biomass Bioenergy. 2015; 74: 135–47. Publisher Full Text\n\nUnderkofler KA, Teixeira RE, Pietsch SA, et al.: Separation of Lignin from Corn Stover Hydrolysate with Quantitative Recovery of Ionic Liquid. ACS Sustain Chem Eng. 2015; 3(4): 606–613. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRahimi A, Ulbrich A, Coon JJ, et al.: Formic-acid-induced depolymerization of oxidized lignin to aromatics. Nature. 2014; 515(7526): 249–252. PubMed Abstract \| Publisher Full Text\n\nPetrasovits LA, McQualter RB, Gebbie LK, et al.: Chemical inhibition of acetyl coenzyme A carboxylase as a strategy to increase polyhydroxybutyrate yields in transgenic sugarcane. Plant Biotechnol J. 2013; 11(9): 1146–1151. PubMed Abstract \| Publisher Full Text\n\nHood EE, Devaiah SP, Fake G, et al.: Manipulating corn germplasm to increase recombinant protein accumulation. Plant Biotechnol J. 2012; 10(1): 20–30. PubMed Abstract \| Publisher Full Text" }	[ { "id": "12515", "date": "17 Feb 2016", "name": "Charles P West", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12516", "date": "17 Feb 2016", "name": "Dominique Loqué", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-185
https://f1000research.com/articles/5-184/v1	17 Feb 16	{ "type": "Review", "title": "Recent advances in dermoscopy", "authors": [ "Teresa Russo", "Vincenzo Piccolo", "Aimilios Lallas", "Giuseppe Argenziano", "Teresa Russo", "Vincenzo Piccolo", "Giuseppe Argenziano" ], "abstract": "The use of dermoscopy has offered a new morphological dimension of skin lesions and has provided an effective diagnostic tool to differentiate melanoma from other benign or malignant skin tumors but also to support the clinical diagnosis in general dermatology. The aim of this article is to provide an overview of the most recent and important advances in the rising world of dermoscopy.", "keywords": [ "Dermoscopy", "melanoma", "basal cell carcinoma", "squamous cell carcinoma", "general dermatology" ], "content": "Introduction\n\nDermoscopy is a non-invasive diagnostic procedure that allows a rapid in vivo evaluation of morphologic structures of the epidermis, the dermoepidermal junction, and the papillary dermis, not visible to the naked eye. Given that dermoscopic structures have been assessed to correlate well with the underlying histopathologic alterations, the method can be regarded as a link between clinical and histopathologic examination. Nowadays, the dermatoscope is considered the dermatologist’s stethoscope and its use has become very popular for both dermatologists and their patients, who often seek dermoscopic examination even when it is unnecessary1,2. Dermoscopy was first introduced to improve melanoma detection, and the evaluation of pigmented and non-pigmented skin tumors still represents its most important indication. However, the continually increasing descriptions of the dermoscopic patterns of several infectious and inflammatory skin diseases steadily establish an essential role for dermoscopy in all fields of dermatology2.\n\nThe beneficial role of dermoscopy in improving melanoma diagnosis has been established at the highest possible level of evidence. However, even after dermoscopic examination, some melanomas might escape detection, either because of their morphologic characteristics or because of the overall patient’s context. In our estimation, the most diagnostically challenging scenarios are the following: melanoma in a patient with multiple moles, slow-growing melanoma (SGM), lentigo maligna (LM), nodular melanoma (NM), and amelanotic melanoma.\n\n\nMelanoma in patients with multiple moles and slow-growing melanoma\n\nRecent evidence confirmed the widespread belief that clinical examination, coupled with dermoscopy, allows the recognition of the majority of melanomas. Specifically, approximately 80% of melanomas are easily recognized on the basis of their clinical or dermoscopic morphologic characteristics, or both. The remaining 20% of melanomas, in contrast, may be missed at the first consultation, since they lack dermoscopic characteristics allowing their discrimination from nevi. The latter is especially relevant in the context of patients with multiple clinically atypical moles, among which melanoma might be perfectly hidden. Effectively, the management of patients with the so-called “atypical mole syndrome” is highly challenging. Excising all (or many) atypical moles is absolutely meaningless, since this strategy induces significant morbidity without reducing at all the risk of melanoma. The optimal strategy for the management of such patients includes total body photography, digital dermoscopic documentation, and periodic monitoring. At the baseline visit, the detailed dermoscopic examination allows the identification of the so-called “signature pattern” of the patient’s nevi, which is based on the observation that the vast majority of an individual’s nevi display similar dermoscopic characteristics. The identification of the patient’s signature nevus pattern is extremely useful because it allows the correct classification of nevi as such while enabling the early recognition of melanoma, which usually deviates from the predominant dermoscopic pattern (ugly duckling or comparative approach). At follow-up visits, clinicians acquire information on the morphologic evolution of lesions with time, which allows the recognition of approximately 10% of melanomas that are morphologically featureless. To optimize the patients’ compliance, the first re-examination should be scheduled at 3 months after the baseline visit, and the following visits at 6- to 12-month intervals. In patients with multiple nevi, a regular annual follow-up is the only safe strategy to detect indolent SGMs characterized by subtle changes over time, which can be recognized only by a prolonged surveillance1,2. A recent study reported on the morphologic evolution of 92 featureless SGMs followed for at least 12 months prior to excision. They have noticed that most of them had minimal (i.e. not more than 2 mm) or no change in size during follow-up but became more disorganized, revealed loss of network in favor of structureless areas, developed a negative network, and exhibited new colors, including dark-brown, black, gray, blue, red, and white3.\n\n\nLentigo maligna\n\nClinical recognition of LM remains one of the most difficult tasks of clinicians, even with the addition of dermoscopy. This diagnostic trouble is related to the fact that LM, pigmented actinic keratosis (PAK), solar lentigo/seborrheic keratosis (SL/SK), and lichenoid keratosis (LPLK) often display overlapping dermoscopic criteria. Comparative studies between LM and SL/SK identified a set of four criteria predictive for LM diagnosis: asymmetric pigmented follicular openings, dark rhomboidal structures, slate-gray globules, and slate-gray dots. In contrast, SL/SK is dermoscopically typified by a sharp demarcation, moth-eaten borders, and fingerprinting. The discrimination between LM and PAK is much more problematic, since the latter has been shown to potentially exhibit all LM criteria. Effectively, a biopsy is often required to differentiate between LM and PAK. Similarly, histopathologic examination represents the only efficient method to classify pigmented facial lesions with extensive regression, where the differential diagnosis lies between regressed melanoma and LPLK (regressed SL/SK). This is because the dermoscopic regression structures of LM and SL/SK are identical structures (gray granules and white areas).\n\nThree simple rules may help to minimize the risk of inappropriate diagnosis and management of LM: (1) the predominance of gray color in facial pigmented macules represents an important alarm sign because it reflects melanin deposition on the upper dermis and within the hair follicles; (2) biopsies of pigmented facial lesions should always be dermoscopy-guided, whereas clinical, dermoscopic, and histopathological findings should always be integrated (i.e. a histological result of a “junctional nevus” on the face of an elderly patient must be surely reviewed); and (3) ablative treatments (e.g. cryotherapy, laser therapy, and so on) should be avoided on equivocal facial lesions4.\n\n\nNodular melanoma\n\nIn contrast to SGM, NM is a rapidly progressing neoplasm that accounts for 10% to 30% of all melanomas and nearly 50% of all melanomas thicker than 2 mm. NM is characterized by a very aggressive biologic behavior, rapidly progressing (or even starting with) a vertical growth phase.\n\nUnfortunately, NM is frequently not diagnosed until progressing to an advanced stage, resulting in a highly unfavorable prognosis. The difficulty in NM recognition results from the fact that it lacks the clinical ABCD criteria (asymmetry, border irregularity, color variegation, and diameter of more than 6 mm), often developing as a perfectly symmetric tumor in terms of shape and color. To address this problem, the “EFG” rule (elevation on cutaneous plane, firmness on palpation, and growth continuous over 1 month) has been introduced in clinical practice to enable the detection of this aggressive melanoma type.\n\nThe recognition of NM is also difficult dermoscopically, since the tumor often lacks the well-known melanoma-specific criteria, whereas available evidence on dermoscopy of NM is relatively scarce. However, during recent years, some studies attempted to test and validate dermoscopic criteria associated with this aggressive melanoma subtype5. Argenziano et al.6 introduced the “blue-back rule”, suggesting that the simultaneous presence of blue and black areas involving at least 10% of the lesion surface each were significantly associated with pigmented NM. Blue color is usually seen as structureless areas, corresponding to aggregations of pigmented melanocytes in the deep dermis. Black color may be seen as dots, globules, or blotches, which result either from superficial (intraepidermal) melanin or from dense dermal proliferations of pigmented melanocytes under a thinned (often because of ulceration) epidermis6. This is in line with the observation that ulceration is more frequent in NM compared with superficial spreading melanoma.\n\nZalaudek et al. suggested that “atypical” vascular structures, including polymorphic vessels, milky red areas, and homogeneous red areas, are also significantly associated with NM7.\n\nIn conclusion, although NM often lacks the “classic” melanoma-specific criteria, dermoscopy might enhance its recognition by revealing blue and black color or abnormal vascular structures or both.\n\n\nAmelanotic and hypomelanotic melanoma\n\nAmelanotic and hypomelanotic melanoma are relatively rare, accounting for less than 2% of all melanomas. Their clinical recognition is particularly difficult, since they might mimic several benign hypopigmented skin lesions, often resulting in a significant delay in diagnosis and deterioration of prognosis. Amelanotic melanoma might develop as a reddish to pinkish macule, papule, plaque, or nodule that rapidly changes in size, shape, and color. Hypopigmented melanoma displays small foci of pigmentation, more frequently located at the periphery of the lesion. Their clinical differential diagnosis includes a variety of benign and malignant lesions8, such as dermal nevus, pyogenic granuloma, adnexal tumor, Spitz nevus, vascular tumors, basal cell carcinoma (BCC), squamous cell carcinoma (SCC), keratoacanthoma, and Merkel cell carcinoma. Since the vast majority of melanoma-associated dermoscopic structures are pigmented, amelanotic melanoma is usually dermoscopically “featureless” and thus difficult to recognize. The most useful dermoscopic criteria are a milky red color and an atypical vascular pattern, consisting of either linear irregular vessels or dotted plus linear vascular structures. Especially in the context of nodular tumors, the only safe strategy not to miss amelanotic melanoma is to excise any lesion that cannot be safely diagnosed as benign after clinical and dermoscopic examination9,10.\n\n\nBasal cell carcinoma\n\nThe value of dermoscopy in improving the diagnosis of BCC has been extensively demonstrated over the last few decades. The most recent advances come from studies suggesting that dermoscopy significantly facilitates the accurate management of the tumor. Specifically, dermoscopy reveals tumor characteristics that might influence the treatment choice, such as the histopathologic subtype and the presence of ulceration or pigmentation.\n\nIn more detail, the dermoscopic criteria associated with non-pigmented BCC include arborizing vessels or fine telangiectasia with few ramifications, ulcerations or multiple small erosions, shiny white-red structureless areas, and short white steaks. The presence of fine telangiectasias with few ramifications or multiple small erosions (or both) predicts the superficial subtype, whereas the presence of arborizing vessels and large ulcerations predicts the nodular subtype.\n\nPigmented BCC is dermoscopically typified by multiple blue-gray dots/globules, in-focus dots, maple leaf-like areas, spoke wheel areas, concentric structures, and blue-gray ovoid nests. The dermoscopic detection of brown-colored structures, including maple leaf-like areas, spoke wheel areas, or concentric structures, is predictive of superficial BCC, whereas the presence of blue-gray ovoid nests predicts a non-superficial subtype.\n\nInfiltrative BCC often exhibits white/red structureless areas, whereas the sclerodermiform BCC often displays a whitish background, corresponding to the underlying fibrosis.\n\nAs described above, dermoscopy provides useful and reliable information on the histopathologic BCC subtype, which is very important for tumor management11. Specifically, when clinical and dermoscopic characteristics are suggestive of superficial BCC, the clinician could consider choosing a non-surgical treatment such as cryotherapy.\n\nIn addition to predicting the histopathologic subtype, dermoscopy might reveal morphologic characteristics of the tumor that are relevant for designing the treatment strategy. For example, the presence of multiple small erosions or ulcerations has been suggested to represent a predictor of favorable response to imiquimod. Another example is the potential of dermoscopy to reveal pigmentation in 30% of clinically non-pigmented BCCs, which is particularly relevant for BCCs scheduled to be treated with photodynamic therapy (PDT). This is because pigmented tumors are known to be less responsive to PDT, since melanin acts as a competitive light-absorbing pigment, thus reducing the tumor’s response rate.\n\nWhen clinical and dermoscopic features of nodular, infiltrative, or sclerodermiform BCC are present, surgical excision undoubtedly represents the first choice to minimize the possibility of tumor recurrence. Moreover, dermoscopy, by providing a more accurate assessment of the true extension of the tumor, allows a more precise estimation of the required surgical margins, helping to minimize the recurrence rates11. In conclusion, dermoscopy not only facilitates the clinical recognition of BCC but also provides additional clues to guide the correct management of the tumor.\n\n\nKeratinocyte skin cancers\n\nThe dermoscopic characteristics of the entities included in the spectrum of keratinocyte skin cancer have been recently investigated. Specific dermoscopic features have been suggested to characterize actinic keratosis (AK), intraepidermal carcinoma (Bowen’s disease), and invasive SCC.\n\nA clinical classification of AKs into three grades has been recently introduced, and different dermoscopic criteria have been suggested to characterize each clinical grade: a red pseudonetwork typifies grade 1 AKs, a strawberry pattern is characteristic of grade 2, and structureless white to yellow areas and keratotic follicular openings are usually seen in grade 3 tumors. Pigmented AKs additionally display a superficial brown network surrounding the follicular openings12.\n\nDermoscopy of non-pigmented intraepidermal carcinoma (Bowen’s disease) reveals glomerular vessels that are arranged in clusters and white to yellow scales. Pigmented Bowen’s disease may also display thick, brown dots with a linear arrangement, usually seen at the periphery of the lesion.\n\nThe dermoscopic pattern of invasive SCC has been shown to depend on the grade of histopathologic differentiation13. In particular, well-differentiated SCC displays signs of keratinization as opaque, yellow scales, a central mass of keratin, structureless white areas, and yellow keratotic follicular plugs surrounded by a white rim (white circle). Linear irregular and, mainly, hairpin vessels might also be seen at the periphery of the tumor, especially in the keratoacanthoma type. In contrast to well- and moderately differentiated SCC, poorly differentiated subtypes commonly lack signs of keratinization, displaying a predominant red color, which results from dense vascularity. Pigmented SCC might reveal a homogeneous pigmentation, irregular blue-gray granular structures, or dark-brown to black crusts when ulcerated11. In conclusion, dermoscopy is useful for diagnosing different stages of keratinocyte skin cancer, improving the optimal tumor management accordingly.\n\n\nDermoscopy in general dermatology\n\nBeyond the well-known value of dermoscopy for the diagnosis of skin tumors, its role in general dermatology is increasingly gaining appreciation among clinical practitioners14–16. The expansion of dermoscopy has been facilitated by the development of handheld polarized dermatoscopes, which are highly portable, do not require skin contact or immersion fluid, and allow fast screening of numerous lesions. Lately, several terms have been suggested to name the use of dermoscopy in different fields, such as trichoscopy for hair disorders, onychoscopy for nail abnormalities, entomodermoscopy for skin infestations, and inflammoscopy for inflammatory skin diseases. Among the several novel applications of dermoscopy, its utility for the diagnosis of inflammatory and infectious skin diseases attracts major interest among dermatologists, given the incidence of these disorders and the difficulties in differential diagnosis in everyday practice. Application of dermoscopy should follow the standard procedure of acquiring information from patient history and clinically evaluating the number, location, and morphology of the lesion(s). Four parameters should be assessed when applying dermoscopy in the realm of inflammatory and infectious diseases—(i) morphological vascular patterns, (ii) arrangement of vascular structures, (iii) colors, and (iv) follicular abnormalities—and the presence of other specific features (clues) should also be evaluated14–16. It must be underlined that dermoscopic findings should always be interpreted within the overall clinical context of the patient, integrated with information from the history and the macroscopic examination. Some dermoscopic criteria appear to be highly specific for a particular disease, whereas others can be seen in more than one entity and subsequently are considered “non-specific”. However, a “non-specific” feature may be rendered particularly valuable when coupled with certain other clinical dermoscopic criteria, forming a set of features that frequently leads to either an accurate single diagnosis or a narrowed list of possible differential diagnoses. Nowadays, common dermatologic diseases can be diagnosed by dermoscopy, which becomes particularly useful in cases of atypical or unusual manifestations. The best-studied disease is psoriasis, which dermoscopically always displays dotted vessels with regular distribution and white scales14–16. Dermoscopy makes differential diagnosis among papulosquamous disorders simpler, permitting clinicians to recognize lichen planus (Wickham striae), eczema (yellow crusts and patchy dotted vessels), and pityriasis rosea (peripheral white scales). Moreover, the detection of a yellow-orange background is considered a dermoscopic clue for the diagnosis of granulomatous skin disorders, such as sarcoidosis, lupus vulgaris, and necrobiosis lipoidica. Dermoscopy has been found useful in the diagnosis of rosacea, discoid erythematous lupus, morphea, lichen sclerosus, pigmented purpuric diseases, Darier’s disease, Grover’s disease, and porokeratosis. In regard to infectious diseases, both frequent (warts, molluscum contagiosum, mycosis, and mite infestations) and uncommon (myiasis and tinea nigra) diseases can be diagnosed by application of dermoscopy, and scabies represents the most striking example (delta-wing jet with contrail sign). Recently, practical tips have been suggested to enhance and optimize the use of dermoscopy by clinicians in their everyday practice of general dermatology. However, given that only a few appropriately designed studies have assessed the diagnostic accuracy of dermoscopy in fields other than skin tumors, these suggestions are based on the expert opinions of a group of clinical and research dermoscopists and should be read with a critical eye, pending further higher-level evidence.\n\n\nConclusions\n\nIn the early years of dermoscopy, the method was considered a second-level tool for further evaluating suspicious skin tumors. With several data and considerable experience gathered in recent decades, the role of dermoscopy became totally different. Today, the dermatoscope is an irreplaceable clinical tool used for the evaluation of virtually every skin lesion, completing the puzzle of clinical examination. Furthermore, the importance of dermoscopy, one of the most popular and dynamic fields of research, in everyday clinical practice is expected to continually increase in the coming years.", "appendix": "Author contributions\n\n\n\nTeresa Russo helped to conceive the review, designed the review, and helped to prepare the first draft of the manuscript. Vincenzo Piccolo helped to conceive the review and to prepare the first draft of the manuscript. Aimilios Lallas and Giuseppe Argenziano helped to conceive the review and 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\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThe authors declare that no grants were involved in supporting this work.\n\n\nReferences\n\nArgenziano G, Ferrara G, Francione S, et al.: Dermoscopy--the ultimate tool for melanoma diagnosis. Semin Cutan Med Surg. 2009; 28(3): 142–148. PubMed Abstract\n\nArgenziano G, Giacomel J, Abramavicus A, et al.: Improving triage and management of patients with skin cancer: challenges and considerations for the future. Expert Rev Anticancer Ther. 2012; 12(5): 609–21. PubMed Abstract \| Publisher Full Text\n\nTerushkin V, Dusza SW, Scope A, et al.: Changes observed in slow-growing melanomas during long-term dermoscopic monitoring. Br J Dermatol. 2012; 166(6): 1213–1220. PubMed Abstract \| Publisher Full Text\n\nLallas A, Argenziano G, Moscarella E, et al.: Diagnosis and management of facial pigmented macules. Clin Dermatol. 2014; 32(1): 94–100. PubMed Abstract \| Publisher Full Text\n\nPizzichetta MA, Kittler H, Stanganelli I, et al.: Pigmented nodular melanoma: the predictive value of dermoscopic features using multivariate analysis. Br J Dermatol. 2015; 173(1): 106–114. 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PubMed Abstract \| Publisher Full Text\n\nLallas A, Apalla Z, Argenziano G, et al.: The dermatoscopic universe of basal cell carcinoma. Dermatol Pract Concept. 2014; 4(3): 11–24. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZalaudek I, Argenziano G: Dermoscopy of actinic keratosis, intraepidermal carcinoma and squamous cell carcinoma. Curr Probl Dermatol. 2015; 46: 70–76. PubMed Abstract \| Publisher Full Text\n\nLallas A, Pyne J, Kyrgidis A, et al.: The clinical and dermoscopic features of invasive cutaneous squamous cell carcinoma depend on the histopathological grade of differentiation. Br J Dermatol. 2015; 172(5): 1308–1315. PubMed Abstract \| Publisher Full Text\n\nLallas A, Giacomel J, Argenziano G, et al.: Dermoscopy in general dermatology: practical tips for the clinician. Br J Dermatol. 2014; 170(3): 514–526. PubMed Abstract \| Publisher Full Text\n\nLallas A, Zalaudek I, Argenziano G, et al.: Dermoscopy in general dermatology. Dermatol Clin. 2013; 31(4): 679–94, x. PubMed Abstract \| Publisher Full Text\n\nZalaudek I, Argenziano G, Di Stefani A, et al.: Dermoscopy in general dermatology. Dermatology. 2006; 212(1): 7–18. PubMed Abstract \| Publisher Full Text" }	[ { "id": "12507", "date": "17 Feb 2016", "name": "Stéphane Dalle", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12508", "date": "17 Feb 2016", "name": "Ralph P. Braun", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-184
https://f1000research.com/articles/5-183/v1	17 Feb 16	{ "type": "Review", "title": "Forest dynamics", "authors": [ "Lee Frelich" ], "abstract": "Forest dynamics encompass changes in stand structure, species composition, and species interactions with disturbance and environment over a range of spatial and temporal scales. For convenience, spatial scale is defined as individual tree, neighborhood, stand, and landscape. Whether a given canopy-leveling disturbance will initiate a sequence of development in structure with little change in composition or initiate an episode of succession depends on a match or mismatch, respectively, with traits of the dominant tree species that allow the species to survive disturbance. When these match, certain species-disturbance type combinations lock in a pattern of stand and landscape dynamics that can persist for several generations of trees; thus, dominant tree species regulate, as well as respond to, disturbance. A complex interaction among tree species, neighborhood effects, disturbance type and severity, landform, and soils determines how stands of differing composition form and the mosaic of stands that compose the landscape. Neighborhood effects (e.g., serotinous seed rain, sprouting, shading, leaf-litter chemistry, and leaf-litter physical properties) operate at small spatial extents of the individual tree and its neighbors but play a central role in forest dynamics by contributing to patch formation at stand scales and dynamics of the entire landscape. Dominance by tree species with neutral to negative neighborhood effects leads to unstable landscape dynamics in disturbance-prone regions, wherein most stands are undergoing succession; stability can only occur under very low-severity disturbance regimes. Dominance by species with positive effects leads to stable landscape dynamics wherein only a small proportion of stands undergo succession at any one time. Positive neighborhood effects are common in temperate and boreal zones, whereas negative effects are more common in tropical climates. Landscapes with positive dynamics have alternate categories of dynamics stabilized by high-severity and low-severity disturbance regimes. Contrary to prevailing ecological theory, systems with positive neighborhood effects can have similar levels of compositional stability across tree, stand, and landscape scales. Neighborhood effect theory can help explain responses of landscapes to large-scale land clearing and novel effects brought on by factors such as invasive species and deer overabundance.", "keywords": [ "Forest dynamics", "Succession", "neighborhood theories", "neighborhood effect", "disturbance dynamics" ], "content": "Introduction\n\nForest dynamics encompass changes in stand structure, species composition, species interactions with disturbance type and severity, and disturbance interactions with landform, over a range of spatial and temporal scales1–4. This article covers forest dynamics in the context of landscape ecology and patch dynamics theory. Forest dynamics as related to biological legacies, land use, herbivory, tree diseases, carbon storage, biogeochemistry, forest health, and climate change are covered elsewhere.\n\nThis review is rooted in classic concepts of landscape ecology, including hierarchical patch dynamics5,6, emergent properties at large spatial extents from interactions among individuals7, resilience, and increasing stability/slower dynamics at landscape than at stand scales5,8,9, as well as the forestry concepts of disturbance severity, stand development, and succession10. However, here neighborhood effects are added to the traditional tree-stand-landscape hierarchy, and stability is examined from the viewpoint of species composition rather than age-class distribution of disturbance patches. This changes the properties of landscape mosaics; four categories of landscape dynamics emerge from a neighborhood effect point of view and they include some seemingly strange elements, such as high stability at small and large spatial extents, and two different types of shifting mosaic steady states. In addition, neighborhood effect theory shows how species not only respond to disturbance but also sometimes regulate disturbance.\n\nThe article starts with a review of forest development and succession, which sets the stage for discussions of species-disturbance interactions, integration of neighborhood effects into cross-scale dynamics, and categories of landscape dynamics. Throughout the article, spatial scales and disturbance severity, as defined in 3, are used. These are individual tree (approximately 0.01 ha), stand, a spatially contiguous collection of trees with similar composition and age structure (0.1 to 10 ha), and landscape, a collection of adjacent stands in the range of 1000 to 100,000 ha. Disturbance severity will have three general categories: low-severity disturbances causing minor mortality in the canopy or understory, or both (e.g., treefall gaps), moderate-severity disturbances causing major mortality of either the overstory or understory (e.g., canopy-leveling wind and moderately severe surface fire), and high-severity disturbances causing major mortality of the overstory and understory (e.g., canopy-leveling wind followed by fire, or high-intensity crown fire). Cumulative disturbance severity includes the additive impacts of multiple disturbances over time that may influence a stand.\n\n\nDevelopment and succession\n\nStand development is directional change in structure over time, whereas succession is directional change in species composition, where early-successional species are replaced by late-successional species3. A sequence of developmental or successional stages can occur by themselves or together. A stand-leveling event such as high-intensity fire, windthrow, or clearcut logging may reset development or succession (or both) to the early stages.\n\nDevelopment occurs in four stages3,10: (1) stand initiation, lasting from the time of canopy disturbance until a new canopy of young trees forms. These are often even-aged stands, or at least even-aged from time of release in cases where advanced regeneration was present. On sites with severe environments, recruitment of new trees may take decades, so that new stands may be unevenly aged by the time of canopy closure. (2) Stem exclusion, lasting from the time of canopy closure through the density-dependent process of self-thinning. (3) Demographic transition (also called understory reinitiation10), begins when tree crowns and resulting gaps formed when trees die are large enough so that trees adjacent to a gap cannot fill them by lateral crown expansion; new cohorts of trees can grow into the overstory. (4) Multi-aged stage (also called old growth10), reached when stage 3 has progressed to the point where only a few individuals from the original even-aged cohort remain, and the canopy has trees of widely varying ages; this stage lasts until another disturbance resets stand development. If the initiation stage was dominated by shade-intolerant, early-successional species, then secondary succession driven by tree species of moderate to high shade tolerance entering the stand may occur along with stand development. However, stand development sequences entirely dominated by late-successional, shade-tolerant species also occur11.\n\nDuring stages 3 and 4 of development, gaps may be filled with small even-aged cohorts of saplings that themselves undergo self-thinning, just like the entire stand did during stages 1 and 2, so that the development process is mirrored in many places at smaller spatial extents. Gap sizes also commonly change from mostly small (50 to 100 m2) in stage 2 and early stage 3 to a wide variety of sizes in late stage 3 and stage 4 (50 to 400 m2); the larger gaps present in these later stages of development lead to opportunities for mid-tolerant species (e.g., Fraxinus, Pinus, Prunus, and Quercus in temperate forests) to maintain or increase their presence in the forest12,13. The light level of the entire stand may be higher in old multi-aged stands, with gaps of widely ranging age and size because side light from numerous gaps can penetrate into the forest around gap edges, so there is no ‘interior zone’ not impacted by gaps14. In addition, partial disturbances that remove 20% to 50% of the canopy, but do not reset the stand to the initiation stage of development, have a large influence on forest dynamics in areas with wind-and-gap types of regimes, and stands hit by such disturbances have relatively high light levels for a few decades after disturbance15.\n\nTraits that determine how a given tree species survives disturbance interact with disturbance type and severity to determine whether a given disturbance will perpetuate the existing species composition or initiate an episode of succession16,17. When disturbance characteristics match survival mechanisms of the dominant tree species then regeneration to pre-disturbance species occurs with little change in composition and development is reset without resetting succession. A mismatch between tree survival mechanisms and disturbance leads to reduction in dominance of pre-disturbance species. Some of the trait-disturbance matching syndromes include (1) species with canopy-stored seed banks, which have several seasons of seeds on hand at any given time, survive disturbance as seeds, and are adapted to high-intensity crown fires on dry sites1, (2) shade-tolerant, late-successional species growing on mesic sites with a seedling/sapling bank in the understory that can respond to overstory wind disturbance18,19, and (3) shade-intolerant or mid-tolerant species with thick bark and foliage held high above the ground, which survive as mature trees adapted to surface fire regimes20. Type 1 forests are maintained at an early-successional stage by fire, and lack of fire or a novel disturbance type (e.g., wind) may never activate the seed bank, leading to recruitment failure and succession to another species. A mismatch for type 2 could be a severe disturbance killing almost all mature trees, seedlings, and seeds. Type 3 forests are usually maintained at a mid-successional stage by fires and could have mismatches caused by a fire that is more severe than normal, killing the mature trees and allowing an earlier-successional species to take over, or lack of fire, allowing late-successional species to dominate21.\n\nSprouting from stumps, root systems, or trunks with broken crowns is a type of survival that is often underappreciated and can allow adaptation to various disturbance types. Crown sprouting, such as occurs in tropical forests after hurricane damage22, is a form of mature-tree persistence that is not common in temperate and boreal forests, although the author has noted that yellow birch (Betula alleghaniensis) and cottonwood (Populus deltoides) are exceptions. Stump and root sprouts are a common form of persistence in aspen (Populus spp.), birch (Betula spp.), and oak (Quercus spp.) species in temperate and boreal forests1,23.\n\nSuccessional status in boreal species can be quite confusing; owing to the low richness of the regenerating species pool, one species can fill more than one successional role. For example, paper birch (Betula papyrifera) and black spruce (Picea mariana) in North America can dominate early post-fire stands as well as old multi-aged stands24,25. This contrasts with progressively higher species turnover across successional gradients as mean annual temperature increases from temperate to tropical forests.\n\nStructural complexity of dead wood and microtopography can influence regeneration dynamics. These biological legacies that persist through, or are formed by, disturbance can have large effects on regeneration dynamics—forests can be ‘born complex’26. Variation in location of large coarse woody debris and pit-and-mound topography can have differential influence on growth and success among regenerating species27, possibly allowing seedlings of some late-successional species to survive after very large, high-severity disturbances.\n\n\nRole of tree species and landform in disturbance\n\nTree species have a surprising ability to regulate disturbance frequency and effects by producing fuel structures at stand and landscape scales that promote high or low fire intensity and rate of spread. Tree susceptibility to wind is a function of wood strength, wood flexibility, tree growth form (tall and thin: susceptible; low center of gravity: less susceptible), and ability to streamline in the wind or to shed branches to reduce pressure caused by wind. Species resistant to wind tend to be late-successional, with some exceptions such as bur oak (Quercus macrocarpa), and arrive at their resistance by various combinations of traits28.\n\nAlthough it is inevitable that any quasi-equilibrium that may exist at the landscape scale will be disrupted at some point, species have a tendency to ‘lock in’ a certain disturbance regime on a certain landform for multiple tree generations—during this time, disturbance and tree traits are in sync—or perhaps out of sync only for a small proportion of stands across the landscape29. Examples (Table 1) include ‘fire pines’ such as jack pine (Pinus banksiana) in the North American boreal forest30,31, growing on dry sites at high canopy densities with repeated high-intensity crown fires at intervals greater than necessary to reach seed-bearing age but shorter than needed for later-successional species to invade. A second example includes eastern hemlock (Tsuga canadensis) and sugar maple (Acer saccharum) that grow on mesic sites not conducive to fire but that reproduce well after treefall events caused by wind at a variety of spatial extents32. A third example is white pine (Pinus strobus) growing on dry-mesic sites with thick bark and low canopy bulk density that favor surface fires in mature stands.\n\nThe examples show species trait, disturbance, and site type combinations that work together to lock in dominance by a given species. The last row also shows disturbance types that are a mismatch and can cause replacement of the dominant species by setting back or advancing succession.\n\nSpecies mosaics can form because of landform-disturbance-tree species interactions, even within one large disturbance patch (Figure 1). This could be caused by spatial variation in disturbance severity associated with landform and also by the fact that different species adapted to the same disturbance type and severity could have differential success on various soil types present within the larger disturbance33. Superimposed on this is additional patch variability caused by the fact that succession is a spatial process; waves of disturbance-sensitive species can move out from surviving populations at disturbance edges and refuges within disturbance perimeters4. Thus, the spatial distribution of landform features at a ‘meso-scale’ (larger than stand but smaller than landscape scale) determines the magnitude of the effects of disturbance size, and landscape resilience to large disturbances2. For fire-sensitive species that can survive fires in swamps or lakeshores, the distribution of such refuge areas across the landscape could cause large variance in the time taken for fire-sensitive, late-successional species to reach stands across the landscape. If one such refuge exists on every 10 ha, for example, then late-successional, fire-sensitive balsam fir (Abies balsamea) could reach the understory of most jack pine forests within several decades, even within a large 100,000-ha fire perimeter. If no such refuges exist, then succession in the middle of a large burn may not take place because of simple lack of availability of late-successional species. In general, refuges from disturbance are frequent on forested landscapes, and overall disturbance size matters much less than one would think by looking at fire perimeter maps. Landscape heterogeneity can provide sites with coarse sandy or rocky soils that serve as permanent seed sources for early-successional species on a landscape otherwise dominated by late-successional species (e.g., white pine in a hemlock-hardwood landscape in northern Wisconsin)32. In addition, landform heterogeneity can divide the landscape into patches (or landscape subsections) with different disturbance regimes, levels of connectivity with other nearby occurrences of the same forest type, and different types of dynamics. In mountainous regions, elevation, aspect, and shape of valleys and ridges create patches that dry out more or less frequently, influencing the frequency, intensity, and direction of movement of fire and wind disturbance, and restricting species movements in response to disturbance. Repeating patterns of lakes, swamps, and rocky ridges can have similar effects in the vast, relatively flat boreal forests of central North America.\n\nNote the central role of neighborhood effects in mediating the relationships among tree species, disturbance, and soil characteristics (blue arrows). Via neighborhood effects, tree species can indirectly influence three of the four factors in stand formation (brown arrows). Stands, combined with landform and disturbance, in turn form the landscape (red arrows).\n\n\nIntegration of dynamics across scales\n\nThe hierarchical tree, stand, and landscape concept has been commonly employed to understand forest dynamics3. At the landscape scale, one can examine the proportion of stands in various stages of succession and development as a result of disturbance history1, as well as proportions of the landscape that may represent different successional pathways due to site type differences—a given landscape is likely to have different successional systems on soils with different water-holding capacity and nutrient status or on subsections of the landscape with different disturbance regimes as mentioned above33,34. At the stand scale, one can examine changes in tree size distributions and species due to development and succession21,35.\n\nA fourth scale, neighborhood (individual trees plus the surrounding neighbors), is also needed to explain forest dynamics. Neighborhood effects are an additional mechanism of patch/stand formation. Trees alter the success of their own regeneration under their own canopy and those of adjacent trees through leaf-litter chemistry and physical characteristics, degree of shade and season(s) of shade cast by a given species, seed rain, sprouting, and disease dynamics36–38. These neighborhood effects can be positive (conspecific regeneration favored), neutral (no effects on abundance of conspecific regeneration), or negative. Neighborhood effects can be mediated by overstory-understory effects within mid- to late-successional forests or disturbance-activated effects such as seed rain from serotinous seeds or sprouting after fire in early-successional forests. It appears that tropical systems have many species with negative neighborhood effects due to disease impacts on seedlings39. These disease feedbacks are uncommon in the temperate zone, although red pine (Pinus resinosa) is an exception40. In temperate and boreal biomes, there are many species whose seedlings do better near conspecific adults, although neutral species are also common41.\n\nWhy are neighborhood effects such an important addition to the traditional tree, stand, and landscape scales? These effects play a central role in mediating the interaction among tree species, soils, and disturbance; they allow tree species to influence three of the four main stand-forming factors (Figure 1). Positive neighborhood effects can cause stands of differing species composition to form on uniform soils in late-successional forests (e.g., hemlock versus sugar maple) and can also cause memory of patch composition through a severe disturbance, as occurs with aspen (root sprouts) versus jack pine (seed rain) patches returning after severe fire3. In effect, strong positive neighborhood effects make the dispersal distance for the pre-disturbance species to recolonize the disturbed area effectively zero—as either seedlings or seeds are present immediately after disturbance throughout a disturbance patch. With positive neighborhood effects, disturbance patches can cross stands dominated by different species, so their boundaries are independent of patch boundaries created by neighborhood effects. Furthermore, patches dominated by different species with positive neighborhood effects act together to influence dynamics of disturbance at larger spatial extents (details below). Thus, neighborhood effects operate at the scale of adjacent trees (0.01 to 0.05 ha) but can create much bigger patches (1 to 10 ha or more) and influence landscape-scale dynamics (Figure 1).\n\nWithout neighborhood effects, all gaps would have random replacement among the species present, and no patches of different species could form on the same soil type independently of disturbance. Neighborhood effects can and do allow coexistence among species with very similar environmental tolerances, via the division of the initially uniform environment into spatially separate patches. Thus, the neighborhood scale is needed to understand patch dynamics, even for locations on uniform soils or within large, high-severity disturbance patches.\n\n\nPositive and neutral/negative neighborhood theories of forest dynamics\n\nThe neighborhood effect theory of forest dynamics was proposed by 3,41. This theory used the cusp catastrophe model42–44 to predict that both continuous and threshold changes in species composition can occur over time. Forests dominated by species with positive neighborhood effects (e.g., serotinous seed rain after fire or a conspecific seedling bank after wind) have a better memory of pre-disturbance composition at neighborhood and stand scales, over a wider range of disturbance severities, than forests dominated by neutral or negative neighborhood species. In essence, predicting forest dynamics without taking neighborhood effects into account amounts to assuming that neighborhood effects are neutral in all cases, and this would therefore be a neutral theory of forest dynamics. Neighborhood effect theory is a more general case that encompasses negative/neutral and positive dynamics.\n\nThe theory also predicted that alternate states of composition (early and late successional) can occur after disturbances of similar severity on landscapes dominated by tree species with positive neighborhood effects, depending on the history of composition. A contrasting continuous response was predicted for forests with neutral neighborhood effects. These predictions were shown to be valid in several case studies41.\n\nThe third major prediction of the theory was the existence of four landscape dynamic categories that emerged from a scaled-up impact of neighborhood effects (Figure 2). The category for a given landscape is related to the portion of the response surface where most of the stands hover, or the portion of the surface to which they are attracted, given the dominant species-disturbance interactions. Category A and B landscapes have high stability across all spatial scales, tree, stand, and landscape because of positive overstory-understory (A) or disturbance-activated neighborhood effects (B). Although prevailing ecological theory recognizes that landscape dynamics can be stabilized by low- or high-frequency disturbance, it does not allow for the substantial degree of stability at smaller spatial extents (e.g., tree and stand) that is allowed for by positive neighborhood effect theory3,9. Other characteristics are mostly opposite; A has mostly late-successional stands and is stabilized by low- to moderate-severity disturbance regimes such as gap dynamics, stand-leveling windthrow, and surface fires, whereas B has mostly early-successional stands and is stabilized by moderate- to high-severity disturbance. Severe disturbance beyond the threshold resets succession in A, whereas in B a lack of disturbance needed to maintain early-successional status allows succession to occur. Note that both A and B landscapes can have mosaics of stands with differing species composition if more than one species with strong positive neighborhood effects is present.\n\nFor positive neighborhood effects, categories A and B represent late- and early-successional landscapes stabilized by low- to moderate-severity wind disturbance regimes and moderate- to high-severity fire regimes, respectively. Some stands may make temporary excursions to the opposite category, but most stands hover on or near the ‘A’ or ‘B’ parts of the response surface. Note that the area of coexistence of early- and late-successional types at moderate disturbance severities depends on history for a given stand (shaded area, left) and that the range of severities that will allow persistence is quite large for A and B. For neutral to negative neighborhood effects, any change in severity over time for D will cause a change in composition as stands slide up and down the response surface. Composition of stands in C can be changed with much less effort than for stands in A; therefore, very stable, low-severity disturbance regimes are required over time to allow C dynamics to persist.\n\nCategory C landscapes have neutral to negative neighborhood effects and low- to moderate-severity disturbance regimes. Tree species can replace each other after small, low-severity disturbances like gap formation, and there is no memory of species composition at individual tree and neighborhood scales. There can be stability at stand to landscape scales for C landscapes, although in temperate and boreal forests, C-type dynamics are very likely to be ephemeral and wiped out by large-scale disturbance or replaced by species with A-type dynamics. However, tropical rain forests, with their negative density-dependent relationships between mature trees and seedling density39 and low chance of fire or other severe disturbance (in the absence of human land-clearing), can likely persist as category C landscapes for centuries, or as long as the climate is stable.\n\nCategory D landscapes are unstable at all spatial scales, as species with neutral neighborhood effects allow composition to change continuously with disturbance severity. A sequence of disturbances of equal severity and uniform temporal spacing would be needed to keep composition the same—an unlikely scenario except perhaps for frequently burned savannas. These are often forests with mixed-severity fire regimes in forest types like Douglas fir (Pseudotsuga menziesii), white pine, and red oak (Quercus rubra), with examples described in 14,18,45.\n\nNote that the traditional steady-state mosaic concept in forest dynamics, defined by 46 as “... an array of irregular patches composed of vegetation of different ages”, did not specify stable or unstable composition at individual tree and neighborhood spatial scales and left the concept vague with respect to steady state within a stand or among stands across a landscape. Neighborhood effect theory divides the steady-state mosaic with gap dynamics as the main disturbance regime into two types of dynamics, A and C, depending on whether tree gap-forming species are replaced with conspecifics or a random assortment of species.\n\n\nNew implications and predictions of neighborhood effect theory\n\nNeighborhood theory predicts the existence of disturbance-mediated accelerated succession47 at stand and landscape scales. If positive disturbance-activated neighborhood effects maintained by fire disappear because of chance absence or exclusion of fire, then late-successional species can enter stands in the understory. A low-severity disturbance regime of gap dynamics or a sudden moderate-severity canopy-leveling windstorm could cause slow or fast transition, respectively, to late-successional species, accompanied by transition from B or D dynamics to A dynamics.\n\nThe theory predicts limitations and (possibly unanticipated) exaggerations of management effects at stand to landscape scales. Landscape dynamics category can be changed by human actions. A very severe disturbance at the landscape scale can override the pre-disturbance memory of the entire landscape and cause a switch from A to B landscape dynamics and initiate widespread succession (as happened when European settlers cleared and burned mesic forest landscapes occupied by sugar maple and hemlock in the Lake States of Michigan, Wisconsin, and Minnesota, USA, in the late 1800s), or the artificial exclusion of severe fire can cause the opposite switch, from B to A (accelerated succession mentioned above or fire suppression in some pine forests21).\n\nThe theory predicts how certain human-induced environmental changes will affect succession and development. Novel ‘disturbances’ and processes may be able to knock out positive neighborhood effects, leading to changed dynamics. For example, the European earthworm invasion is resulting in loss of the organic horizon in North American forests48, possibly removing neighborhood effects that operate through leaf-litter effects, while overabundant deer could eliminate the seedling layer49 that is crucial to positive overstory-understory effects. Either of these could destabilize the hemlock and maple mosaics common in eastern North America that were maintained by positive neighborhood effects.\n\n\nConclusions\n\nTree species have a surprising level of control over disturbance dynamics because some tree species can ‘lock in’ disturbance and species interactions on certain landforms to create periods of stability in composition at the landscape scale41, which may end when a large infrequent disturbance occurs17. However, the basic tree-trait and survival mechanism interactions with disturbance as an arbiter of whether the pre-disturbance species will persist after disturbance, or whether succession will be reset, need to be reassessed in light of the cross-scale impacts of neighborhood effects and landform effects. Landform and soil-type effects can create mosaics of stand types within single disturbance patches, whereas neighborhood effects can cause different stand types to separate even within one uniform soil type, and neighborhood effects also cause interactions of groups of positive or neutral species with disturbance to form different categories of landscape dynamics. Thus, the tree-stand-landscape hierarchy is not as straightforward as it may first appear because of the complex cross-linkages among levels and ability of tree species to extend their influence very strongly to stand and landscape scales via neighborhood effects that modify disturbance dynamics (Figure 1). From an ecological process and disturbance dynamics perspective, the hierarchy does not nest as neatly as it may from a spatial patch dynamics perspective. A complete theory integrating all of the elements in Figure 1 awaits development.\n\nUnfortunately, not enough is known to parameterize the neighborhood effect cusp catastrophe model and predict the actual threshold disturbance severity for a state change, and this is due to difficulties in developing universal measures of disturbance severity and neighborhood effect strength. For now, the theory can be used in a semi-quantitative sense with disturbance severity and neighborhood effect categories. A breakthrough is needed in understanding of disturbance severity and how to measure it, as well as disturbance severity versus disturbance type effects, which are frequently confounded.", "appendix": "Competing interests\n\n\n\nThe author declares that he has 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\nJohnson EA: Fire and vegetation dynamics: studies from the North American boreal forest. Cambridge University Press, Cambridge, England. 1992. Reference Source\n\nTurner MG, Romme WH, Gardner RH, et al.: Effects of fire size and pattern on early succession in Yellowstone National Park. Ecol Monogr. 1997; 67(4): 411–433. Publisher Full Text\n\nFrelich LE: Forest Dynamics and disturbance regimes. Cambridge University Press, Cambridge, England. 2002. 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Publisher Full Text \| Faculty Opinions Recommendation\n\nHanson JJ, Lorimer CG: Forest structure and light regimes following moderate wind storms: implications for multi-cohort management. Ecol Appl. 2007; 17(5): 1325–1340. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nWhelan RJ: The ecology of fire. Cambridge University Press, Cambridge, England. 1995. Reference Source\n\nTurner MG, Baker WL, Peterson CJ, et al.: Factors influencing succession: Lessons from large, infrequent natural disturbances. Ecosystems. 1998; 1(6): 511–23. Publisher Full Text\n\nHough AF, Forbes RD: The ecology and silvics of forest in the high plateaus of Pennsylvania. Ecol Monogr. 1943; 13(3): 299–320. Reference Source\n\nRunkle JR: Gap regeneration in some old-growth mesic forests of eastern North America. Ecology. 1981; 62(4): 1041–1051. Reference Source\n\nHeinselman ML: The Boundary waters wilderness ecosystem. Minneapolis, Minnesota, The University of Minnesota Press. 1996. 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Publisher Full Text \| Faculty Opinions Recommendation\n\nDonato DC, Campbell JL, Franklin JF: Multiple successional pathways and precocity in forest development: can some forests be born complex? J Veg Sci. 2012; 23(3): 576–584. Publisher Full Text \| Faculty Opinions Recommendation\n\nVodde F, Jogiste K, Engelhart J, et al.: Impact of wind-induced microsites and disturbance severity on tree regeneration patterns: Results from the first post-storm decade. Forest Ecol Manag. 2015; 348: 174–185. Publisher Full Text\n\nFrelich LE, Ostuno EJ: Estimating wind speeds of convective storms from tree damage. Electronic J Severe Storms Meteor. 2012; 7(9): 1–19. Reference Source\n\nKeeley JE, Pausas JG, Rundel PW, et al.: Fire as an evolutionary pressure shaping plant traits. Trends Plant Sci. 2011; 16(8): 406–411. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nMutch RW: Wildland fires and ecosystems: a hypothesis. Ecology. 1970; 51(6): 1046–1051. Reference Source\n\nKeeley JE, Zedler PH: Evolution of life histories in Pinus. D.M. Richardson, editor, Ecology and biogeography of Pinus. Cambridge University Press, Cambridge, England. 1998; 219–250. Reference Source\n\nFahey RT, Lorimer CG, Mladenoff DJ: Habitat heterogeneity and life-history traits influence presettlement distributions of early-successional tree species in a late-successional, hemlock-hardwood landscape. Landscape Ecol. 2012; 27(7): 999–1013. Publisher Full Text \| Faculty Opinions Recommendation\n\nBridge SRJ, Johnson EA: Geomorphic principles of terrain organization and vegetation gradients. J Veg Sci. 2000; 11(1): 57–70. Publisher Full Text \| Faculty Opinions Recommendation\n\nLorimer CG: The presettlement forest and natural disturbance cycle of northeastern Maine. Ecology. 1977; 58(1): 139–148. Reference Source\n\nLorimer CG: Age structure and disturbance history of a southern Appalachian virgin forest. Ecology. 1980; 61(5): 1169–1184. Reference Source\n\nFerrari JB: Fine-scale patterns of leaf litterfall and nitrogen cycling in an old-growth forest. Can J Forest Res. 1999; 29(3): 291–302. Publisher Full Text\n\nRothe A, Binkley D: Nutritional interactions in mixed species forests: a synthesis. Can J Forest Res. 2001; 31(11): 1855–1870. Publisher Full Text \| Faculty Opinions Recommendation\n\nCanham CD, LePage PT, Coates KD: A neighborhood analysis of canopy tree competition: effects of shading versus crowding. Can J Forest Res. 2004; 34(4): 778–787. Publisher Full Text \| Faculty Opinions Recommendation\n\nMangan SA, Schnitzer SA, Herre EA, et al.: Negative plant-soil feedback predicts tree-species relative abundance in a tropical forest. Nature. 2010; 466(7307): 752–755. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nHaugen LM, Ostry ME: Long-term impact of shoot blight disease on red pine saplings. North J Appl For. 2013; 30(4): 170–174. Publisher Full Text \| Faculty Opinions Recommendation\n\nFrelich LE, Reich PB: Minireviews: Neighborhood effects, disturbance severity, and community stability in forests. Ecosystems. 1999; 2(2): 151–166. Publisher Full Text\n\nZeeman EC: Catastrophe theory. Sci Am. 1976; 234(4): 65–83. Publisher Full Text\n\nHolling CS: Forest insects, forest fires, and resilience. In Fire regimes and ecosystem properties. USDA Forest Service Technical Report WO-26. 1981; 445–464. Reference Source\n\nLoehle C: Catastrophe theory in ecology: a critical review and an example of the butterfly catastrophe. Ecol Modell. 1989; 49(1–2): 125–152. Publisher Full Text\n\nAbrams MD: Fire and the development of oak forests: In eastern North America, oak distribution reflects a variety of ecological paths and disturbance conditions. Bioscience. 1992; 42(5): 346–353. Publisher Full Text\n\nBorman FH, Likens GE: Pattern and Process in a Forested Ecosystem. Spinger-Verlag, New York. 1979.\n\nAbrams MD, Scott ML: Disturbance-mediated accelerated succession in two Michigan forest types. Forest Sci. 1989; 35(1): 42–49. Reference Source\n\nBohlen PJ, Scheu S, Hale CM, et al.: Non-native invasive earthworms as agents of change in northern temperate forests. Front Ecol Environ. 2004; 2(8): 427–435. Publisher Full Text \| Faculty Opinions Recommendation\n\nCôté SD, Rooney TP, Tremblay JP, et al.: Ecological impacts of deer overabundance. Annu Rev Ecol Syst. 2004; 35(1): 113–147. Publisher Full Text \| Faculty Opinions Recommendation" }	[ { "id": "12489", "date": "17 Feb 2016", "name": "Edward Johnson", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12488", "date": "17 Feb 2016", "name": "Klaus Puettmann", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-183
https://f1000research.com/articles/5-182/v1	17 Feb 16	{ "type": "Review", "title": "Single-cell transcriptome sequencing: recent advances and remaining challenges", "authors": [ "Serena Liu", "Cole Trapnell", "Serena Liu" ], "abstract": "Single-cell RNA-sequencing methods are now robust and economically practical and are becoming a powerful tool for high-throughput, high-resolution transcriptomic analysis of cell states and dynamics. Single-cell approaches circumvent the averaging artifacts associated with traditional bulk population data, yielding new insights into the cellular diversity underlying superficially homogeneous populations. Thus far, single-cell RNA-sequencing has already shown great effectiveness in unraveling complex cell populations, reconstructing developmental trajectories, and modeling transcriptional dynamics. Ongoing technical improvements to single-cell RNA-sequencing throughput and sensitivity, the development of more sophisticated analytical frameworks for single-cell data, and an increasing array of complementary single-cell assays all promise to expand the usefulness and potential applications of single-cell transcriptomic profiling.", "keywords": [ "Single-cell RNA-sequencing", "single-cell transcriptomic profiling" ], "content": "Introduction\n\nThe advent of next-generation sequencing over a decade ago spurred the development of a host of sequencing-based technologies1 for probing genomic variation and dynamics. Of these methods, RNA-sequencing (RNA-seq) enabled transcriptomic profiling at unprecedented sensitivity and breadth, leading to the discovery of new RNA species and deepening our understanding of transcriptome dynamics2,3. In recent years, low-input RNA-seq methods have been adapted to work in single cells4. These single-cell RNA-seq (scRNA-seq) technologies can quantify intra-population heterogeneity and enable study of cell states and transitions at very high resolution, potentially revealing cell subtypes or gene expression dynamics that are masked in bulk, population-averaged measurements5,6. In this review, we will discuss recent advancements and current limitations of scRNA-seq methodologies and highlight major applications of scRNA-seq in biological research.\n\n\nscRNA-seq technologies: overview and recent advancements\n\nOver the past six years, numerous scRNA-seq protocols have been developed4,7–21. Currently published scRNA-seq protocols all follow the same general workflow: single cells are isolated; cells are lysed, and the RNA is captured for reverse transcription into cDNA; and the cDNA is pre-amplified and then used to prepare libraries for sequencing and downstream analysis. Kolodziejczyk et al.22 provide a comprehensive review of individual scRNA-seq protocols and their relative strengths and weaknesses.\n\nAlthough cDNA pre-amplification is necessary because only minute amounts of RNA are captured from each cell23, amplification bias arising during pre-amplification limits the quantitative accuracy of scRNA-seq. Unique molecular identifiers (UMIs) can be used to barcode individual RNA molecules during the reverse transcription step, allowing direct transcript counting24–29, and many of the newer scRNA-seq protocols use UMIs to improve transcript quantitation9,16–19. Alternatively, exogenous RNA standards such as those from the External RNA Control Consortium (ERCC) can be “spiked in” with cellular RNA to map between relative and absolute transcript counts20,30. Stegle et al.31 provide a more detailed discussion of methods for scRNA-seq transcript quantitation and highlight some of the analytical challenges unique to single-cell data.\n\nscRNA-seq methods have also been improving in terms of throughput and scalability. Whereas most earlier methods have been limited to measuring hundreds or thousands of cells at a time, recent advancements in microwell17 and droplet-based18,19 cell-barcoding strategies have enabled the analysis of tens of thousands of cells in a single experiment. The high-throughput capacity of these new technologies will increase the resolution of single-cell experiments, improving their ability to detect rare cell subtypes or transitional states.\n\n\nChallenges and limitations of scRNA-seq\n\nCurrent scRNA-seq technologies still face a number of challenges. Collectively, existing scRNA-seq methods have low capture efficiency. Because only a small fraction of each cell’s transcript complement (approximately 10% for many protocols9) is represented in the final sequencing libraries, scRNA-seq has limited sensitivity and is unable to reliably detect low-abundance transcripts9,32,33. The low amount of input material for scRNA-seq libraries also leads to high levels of technical noise, which complicates data analysis and can mask underlying biological variation22,34–37. Methods for modeling technical variation in scRNA-seq data have been proposed35–37; however, most approaches use the sample-to-sample variation in ERCC read counts to model and control for technical noise in the single-cell data and thus can be used only with experiments incorporating spike-in controls. Moreover, these approaches assume that the spike-in transcripts are treated the same as cellular RNA during library prep. However, naked spike-in RNA does not pass through cellular lysis and is not in complex with ribosomes or RNA-binding proteins. Thus, although spike-in procedures serve as useful indicators of transcript frequency and sensitivity in an experiment, there are many sources of variability that remain difficult to control in scRNA-seq.\n\nAnother potential source of bias stems from procedures to isolate and capture individual cells. Although micromanipulation or laser dissection techniques can isolate single cells from known locations within a cell population or tissue, these methods are labor-intensive or require specialized equipment22,33,38. Most scRNA-seq protocols—and all of the existing high-throughput methods—first dissociate tissues to form a single-cell suspension before capturing individual cells. This cell dissociation step is often non-trivial, and enzymatic treatments used to break down tissues may impact cell viability, potentially affecting cells’ transcriptional profiles22. To avoid biases stemming from such enzymatic treatments, Grindberg et al. have developed techniques for performing RNA-seq directly on single nuclei39,40, which can be isolated without using harsh protease treatments.\n\nFor most single-cell isolation procedures, information about cells’ original spatial context and cellular environment is lost. Recently, computational methods have been developed to infer a cell’s original position in three-dimensional space from its transcriptional profile by using a reference gene expression map built from existing in situ data41,42. However, these methods rely on the existence of spatial expression data for a panel of reference genes in the tissue of interest. Alternatively, emerging in situ sequencing strategies are able to capture and amplify RNA within the original tissue context, although current methods can measure up to only a few dozen genes per cell43–45. These methods sequence RNA directly inside unlysed cells: cDNA amplicons are generated and circularized, amplified via rolling circle amplification, and then sequenced by ligation in situ by using the SOLiD platform44,45. Such in situ sequencing approaches are distinct from fluorescence in situ hybridization (FISH) strategies (discussed further below), which detect transcripts through the binding of fluorescently labeled probes. However, although in situ sequencing methods preserve spatial information and can measure RNA expression patterns at subcellular resolution, these approaches are currently limited in throughput and require specialized tools which may not be widely accessible.\n\nFinally, the bulk of scRNA-seq literature has focused solely on polyadenylated mRNAs; almost all published scRNA-seq protocols isolate cellular RNA by using poly-T priming, which captures only polyadenylated transcripts. Consequently, current methods are ill suited to investigate non-polyadenylated transcript classes, such as regulatory non-coding RNA (e.g. microRNAs46,47, lncRNAs48, or circular RNAs49,50) or bacterial RNA21. Random hexamer priming has been suggested as a strategy to simultaneously capture both polyadenylated and non-polyadenylated transcripts in single cells20,21, and computationally selected “not-so-random” primers could potentially be used to capture poly(A)+ and poly(A)– species while depleting for ribosomal RNA51. Incorporating these alternative priming strategies into existing scRNA-seq technologies would enable the exploration of a wider spectrum of transcript types, broadening the scope and applicability of scRNA-seq.\n\n\nComplementary single-cell technologies\n\nAlthough scRNA-seq alone is a powerful tool for dissecting cell populations and processes, combining scRNA-seq with other single-cell technologies supplements transcriptomic data with complementary information that helps to paint a more complete picture of each cell. RNA FISH, in which individual transcripts are labeled with fluorescent probes and then detected via high-resolution microscopy, provides an orthogonal method of quantifying transcript levels and is often used to independently validate results from scRNA-seq data52. Unlike scRNA-seq, single-cell FISH preserves the spatial context of assayed transcripts and can localize molecules down to subcellular resolution53,54. RNA localization and trafficking dynamics often play a crucial role in regulating protein translation and cellular function55; used in conjunction with scRNA-seq, single-cell FISH could supplement the global transcriptomic snapshots of scRNA-seq with information on the spatial dynamics of selected transcripts. Whereas spectral overlap between fluorophores still limits the number of transcripts that can be simultaneously assayed, new approaches using super-resolution microscopy and combinatorial labeling schemes can measure up to thousands of transcripts in each cell53,54,56.\n\nSingle-cell genome sequencing has been developing alongside scRNA-seq and has been used successfully to map genetic variation at single-cell resolution and to infer cell lineages57–61. Moreover, in the past year, methods have been developed to sequence both the genome and the transcriptome of the same cell62,63, enabling direct comparison of genetic and gene expression variation within a single cell. This integrated, parallel-sequencing approach shows great promise for uncovering genotype-phenotype relationships and has already been used to demonstrate strong correlations between gene copy number and gene expression levels62,63.\n\nOver the past few years, methods have also been developed to assay the epigenetic landscape of single cells: both bisulfite sequencing64–67 (measuring DNA methylation) and assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq)68,69 (measuring chromatin accessibility) have been adapted to work with single cells. These methods offer insight into the epigenetic heterogeneity within cell populations, and paired epigenomic and transcriptomic data could deepen our understanding of the mechanisms underlying gene expression regulation. Although direct comparison of a cell’s epigenomic and transcriptomic profiles is not currently possible, combining single-cell bisulfite sequencing or single-cell ATAC-seq with scRNA-seq from the same cell could enable such analyses in the future. Similarly, integrating scRNA-seq with single-cell proteomic methods70,71 would provide insight into post-transcriptional gene regulation and the degree to which mRNA expression is reflected at the protein level.\n\n\nApplications of scRNA-seq\n\nRecent studies have demonstrated high cell-to-cell transcriptomic variation10,72–74, even within genetically homogenous cell populations75. Consequently, bulk measurements can mask important cellular heterogeneity5,76 and lead to averaging artifacts6. One major advantage of scRNA-seq is its ability to detect such cell-to-cell heterogeneity and capitalize upon it to uncover population structure and cell dynamics hidden at the group level.\n\nscRNA-seq has been used to dissect heterogeneous cell populations and complex tissues, such as intestine77, spleen16, lung78, or brain42,79–83. Clustering methods16,75,77 or dimensionality reduction techniques78 can be used directly on single-cell expression data to group cells by transcriptomic similarity and to detect the underlying population structure in an unsupervised manner (Figure 1A). Cell subgroups identified from such analyses can often be matched to known cell types via previously established marker genes16,52,78,81,82; however, structural analysis of single-cell data has also led to the discovery of novel cell subtypes79,83,84 as well as the identification of new marker genes for known cell types78,84,85. In the context of cancer, scRNA-seq analyses have been used to characterize intra-tumoral heterogeneity and to classify tumor subpopulations86–88. scRNA-seq profiling can also detect variation among cell states within a seemingly homogenous population, such as differences in cell cycle stage89 or differential signaling responses to an outside stimulus52,75,90.\n\n(a) Deconvolving heterogeneous cell populations. Clustering by single-cell transcriptomic profiles can reveal population substructure and enable the identification of cell subtypes and rare cell species (e.g. red cells above). Clusters may be tight and well defined (purple, red) or diffuse (blue). (b) Trajectory analysis of cell state transitions. Single-cell RNA sequencing time-series data can be used to map cell developmental trajectories over the course of dynamic processes such as differentiation or signaling responses to an external stimulus. Some computational suites (e.g. Monocle6) can also accommodate branching trajectories, enabling identification of lineage-specific gene expression and key genes that drive branching events. (c) Dissecting transcription mechanics. Genes’ expression profiles across many cells can be compared to study transcriptional bursting and to model the kinetics of stochastic gene expression. (d) Network inference. Genes can be clustered by expression profile to identify modules of putatively co-regulated genes, and gene-gene covariation relationships can be used to infer gene regulatory networks or subnetworks.\n\nscRNA-seq is also commonly used to study cellular transitions between different states and to map cell trajectories through processes like differentiation (Figure 1B). Several analytical frameworks have been proposed for inferring such trajectories: Monocle introduced the concept of “pseudotime” as a quantitative measure of “progress through a biological process” and uses techniques from computational geometry to order cells in pseudotime on the basis of their transcriptomic profiles6. Wanderlust uses an entirely different algorithm based on local topological clustering to place cells along a developmental trajectory91 by using single-cell proteomic measurements. More recently, Shin et al.92 and Moignard et al.93 have outlined additional strategies for reconstructing cell trajectories. Once cells have been ordered along a trajectory, gene expression patterns over the course of the established developmental trajectory can be analyzed to identify key regulators and genes with “switch-like” behavior6,72,91. Sensitivity for identifying intermediate differentiation states can also be improved by using latent variable models to account for potential confounding factors (such as cell cycle) in the expression data prior to applying trajectory analysis techniques94.\n\nGrowing evidence suggests that genes are not transcribed continuously but rather undergo short bursts of transcription interspersed with silent intervals95. Transitions between “on” and “off” states are governed by several stochastic processes96,97, and this phenomenon of “transcriptional bursting” is a major source of gene expression heterogeneity between cells. scRNA-seq can be used to explore transcriptional mechanics and to model the kinetics of stochastic gene transcription96,98,99 (Figure 1C). Recent studies have also reported instances of cells preferentially expressing a single allele32 or a single splice isoform75; however, the low mRNA capture efficiency of scRNA-seq makes it difficult to draw definitive conclusions about allele-specific or isoform-specific expression at the single-cell level.\n\nThe inherent gene expression variability between cells in scRNA-seq data can be used to infer gene regulatory networks (GRNs)100–102. Most commonly, genes are grouped into co-regulated “modules” on the basis of expression profile similarity16,52,75,86,87,103 (Figure 1D). Network inference from scRNA-seq data poses several challenges. Owing to low capture efficiency and stochastic gene expression, gene dropout (where gene expression is zero in a given cell) is quite common, leading to zero-inflated expression data104. Although zero-inflated distributions can be used to accommodate expected dropout104–106, such models also have a greater number of parameters and can be more difficult to fit than a simpler model, particularly when sample size is limited. As previously mentioned, scRNA-seq data are very noisy, and separating biological variation from technical noise remains a non-trivial problem35,36. Additionally, the number of model parameters to be estimated (genes and gene interactions) usually greatly exceeds the number of sample observations (cells measured), and this disparity poses challenges for parameter estimation107,108. Simplifying the model on the basis of prior knowledge or focusing on only a small subnetwork of key players may be necessary to make parameter estimation feasible107–110. Finally, experimentally validating inferred GRNs can be very difficult; whereas knocking out a single gene is relatively straightforward, disrupting interactions between two proteins or between a protein and its target sequence can be much harder, and very few hypothesized models have been rigorously tested thus far.\n\n\nConclusions\n\nscRNA-seq technologies have advanced significantly since their inception, improving in terms of both transcript quantitation and experimental throughput. Whereas low capture efficiency and high levels of technical noise limit the sensitivity and accuracy of scRNA-seq, more sophisticated analytical frameworks are emerging to facilitate the interpretation of scRNA-seq data35–37. Pairing single-cell transcriptomic data with spatial information41,42,54 or orthogonal single-cell genomic assays62,63,65,68 also promises to provide new insights into transcriptional dynamics and the mechanisms underlying gene regulation.\n\nscRNA-seq has been very effective at dissecting complex, heterogeneous cell populations, enabling unsupervised learning of population structure and the discovery of novel subtypes and rare cell species79,84. In the context of dynamic processes, cell trajectories reconstructed from single-cell transcriptomic data have provided insight into transient intermediate cell states and have helped to identify key regulator genes6,91. Finally, scRNA-seq also shows great potential for elucidating stochastic transcriptional kinetics and inferring gene regulatory networks. However, network inference from scRNA-seq data is computationally challenging and difficult to validate; inferred network models should thus be critically evaluated and experimentally tested where possible.", "appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nCT is supported by an NIH DP2 HD088158 grant, an Alfred P. Sloan Fellowship, and a Dale F. Frey Award for Breakthrough Scientists from the Damon Runyon Cancer Research Foundation. SL is supported by an NSF IGERT grant DGE-1258485.\n\n\nReferences\n\nSoon WW, Hariharan M, Snyder MP: High-throughput sequencing for biology and medicine. Mol Syst Biol. 2013; 9: 640. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang Z, Gerstein M, Snyder M: RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet. 2009; 10(1): 57–63. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "12513", "date": "17 Feb 2016", "name": "Sten Linnarsson", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12514", "date": "17 Feb 2016", "name": "Roger S Lasken", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-182
https://f1000research.com/articles/5-180/v1	16 Feb 16	{ "type": "Research Note", "title": "Analysis of red autofluorescence (650-670nm) in epidermal cell populations and its potential for distinguishing contributors to 'touch' biological samples", "authors": [ "Cristina E. Stanciu", "M. Katherine Philpott", "Eduardo E. Bustamante", "Ye Jin Kwon", "Christopher J. Ehrhardt", "Cristina E. Stanciu", "M. Katherine Philpott", "Eduardo E. Bustamante", "Ye Jin Kwon" ], "abstract": "Interpretation of touch DNA mixtures poses a significant challenge for forensic caseworking laboratories. Front end techniques that facilitate separation of contributor cell populations before DNA extraction are a way to circumvent this problem. The goal of this study was to survey intrinsic fluorescence of epidermal cells collected from touch surfaces and investigate whether this property could potentially be used to discriminate between contributor cell populations in a biological mixture. Analysis of red autofluorescence (650-670nm) showed that some contributors could be distinguished on this basis. Variation was also observed between autofluorescence profiles of epidermal cell populations from a single contributor sampled on different days. This dataset suggests that red autofluorescence may be a useful marker for identifying distinct cell populations in some mixtures. Future efforts should continue to investigate the extrinsic or intrinsic factors contributing to this signature, and to identify additional biomarkers that could complement this system.", "keywords": [ "forensic science", "flow cytometry", "epidermal cell", "touch DNA", "autofluorescence", "mixture" ], "content": "Introduction\n\nThe difficulties associated with interpreting complex DNA mixtures are well known in the forensic community, and are becoming more prevalent with the sharp increase in ‘touch’ or trace samples among forensic laboratories’ caseloads1. Differentiating cell populations from individual contributors in a biological mixture before DNA analysis is a potential way to overcome this issue. While strategies exist to selectively label cell populations from distinct contributors based on their immunochemistry and then physically isolate cells from the mixture prior to DNA profiling2–4, there is a dearth of studies demonstrating cell separation techniques on touch samples. This is likely due to the fact that cell populations in these samples mostly, if not entirely, consist of fully differentiated keratinocytes which have limited reactivity to common molecular probes used to target surface antigens5,6.\n\nAn alternative approach is to avoid the need for probe binding by harnessing the intrinsic fluorescence of compounds found in or on epidermal cells. Here we report on our analysis of autofluorescence in the red region of the spectrum (650–670nm) of epidermal cells collected from surfaces touched by seven different individuals across multiple days, and the implications this may have for processing complex biological mixtures in forensic casework.\n\n\nMethods\n\nTouch samples were collected from seven volunteers using the following protocol which was approved by the VCU-IRB (#HM20000454_CR). Volunteers rubbed a sterile polypropylene conical tube (P/N 229421; Celltreat Scientific) for five minutes using their entire hand (i.e., palm and fingers). Cells were collected from the surface with six sterile pre-wetted swabs (P/N 22037924; Fisher Scientific) followed by two dry swabs. To elute the cells into solution, the swabs were manually stirred then vortexed for 15 seconds in 10 mL of ultrapure water (18.2 MΩ∙cm). The entire solution was then passed through a 100 µm filter mesh prior to flow cytometry. Flow cytometry analysis of eluted cells was performed on the BD FACSCanto™ II Analyzer (Becton Dickinson) equipped with 488 nm and 633 nm lasers and a 660/20 nm detector filter. Channel voltages were set as follows: Forward Scatter (FSC, 150V), Side Scatter (SSC, 200V) and Allophycocyanin (APC, 250V). FSC and SSC channels were used to gate intact corneocytes for subsequent autofluorescence analysis. Gating of cell populations and generation of histogram profiles for each contributor was performed using FCS Express 4.0 Flow Research Edition (De Novo Software, Inc.).\n\n\nResults and discussion\n\nFluorescence histograms of individual cell populations from different donors are shown in Figure 1. For ease of comparison and visualization, profiles have been overlayed and grouped by the day on which cells were deposited, collected, and analyzed by flow cytometry. Clear differences in the red fluorescence (APC) channel are observed between several pairs of donor cell populations, particularly J16-D02 during the first experiment and J16-S07 in the second experiment (Figures 1a and 1b respectively; Table 1). Most experiments resulted in one or more contributor cell population(s) whose fluorescence profile(s) could be distinguished from the others collected that day, such that a fluorescence intensity gate could be designed that would be expected to capture that contributor’s cells to the exclusion of (or minimal contribution of) cells from other contributors. However, significant and/or complete overlap was observed between many donor pairs (e.g., A42-B17 in Figure 1a; I66-S07 in Figure 1d). Sometimes, overlap of fluorescence distributions was such that gating could potentially separate the contributors into two or more groups (e.g. Figure 1d: A42, B17, I66, R12 and S07 in one group; D02 and J16 in another group). All contributors from the final experiment exhibited overlapping fluorescence histograms (Figure 1e).\n\nPanels a–e show different combinations of donors cell populations each sampled and analyzed on the same day. Figure 1f is a histogram overlay of cell populations from contributor J16 across five different experiments.\n\n1Data is organized according to the histogram overlays shown in Figure 1. Mean (arithmetic) and median values are in relative fluorescent units (RFUs).\n\n2Flow cytometry cell ‘events’ correspond to populations within FSC and SSC gates that select for intact epidermal cells.\n\nCell populations from J16 and D02 showed a great deal of disparity in fluorescence intensity in the first experiment, such that overlap between these populations was minimal (Figure 1a). There was somewhat less distinction – and thus more overlap – observed between the same contributors during a second replicate (Figure 1c); during a third, overlap between the two populations was substantial (Figure 1d). As these results suggest, fluorescence intensity values for cell populations derived from any given contributor varied in distribution across replicate experiments on different days. Figure 1f shows overlayed histograms for J16 cell populations; mean fluorescence intensity values ranged from 589 to 2606 relative fluorescence units (RFUs) across five sampling days (Table 1).\n\nThe underlying cause of red autofluorescence in these epidermal cell samples is currently unclear. Cells deposited through touch are likely primarily derived from the outermost epidermal layer (stratum corneum) which can contain a number of fluorescent compounds including tryptophan and tyrosine7,8, melanin, keratins, NADH and flavins9, lipofuscins10, and porphyrins and porphyrin precursors11,12. However, many of the corresponding emission maxima for these molecules occur at shorter wavelengths than what was examined in this study (e.g., amino acids, keratin, NADH, all have maxima below 550nm9). Porphyrin molecules exhibit emission maxima between 630–680nm11. Their abundance within the epidermis may be influenced by bacteria on the skin that produce porphyrin molecules with similar fluorescence emission profiles13. Exogenous sources such as plasticides14 or other biological compounds (e.g., chlorophyll15) may also produce fluorescence, and could potentially be transferred to donors’ hands and subsequently to the tube surface (with cells) through touch or contact.\n\nRegardless of the ultimate source for the observed differences in cell population fluorescence, this initial data set indicates that autofluorescence may be a useful marker for distinguishing between cell populations in a mixture. The non-destructive nature of flow analysis and the fact that autofluorescence monitoring does not require special reagents beyond those maintained in any laboratory (e.g. no probes required) are advantages when considering their potential front-end use in forensic analyses.\n\nThe variation across multiple samples from the same donor suggests that the level of autofluorescence is likely not a unique or identifying feature for a particular individual. However, to be of use in separating components of a biological mixture, a feature need not be unique; it simply needs to be distinctive among the contributors to that particular mixture. The ability to separate out even one contributor (or to separate a mixture of four contributors into two mixtures of two) may render the remaining mixture more interpretable in downstream DNA analysis. Further, the possibility that some combination of endogenous and/or exogenous factors could impart distinct optical properties to contributor cell populations in a particular mixture sample warrants further exploration.\n\nFuture efforts will continue to focus on isolating the molecule(s) responsible for fluorescent differences in touch epidermal cells through a combination of targeted immunofluorescent assays, chemical characterizations, and complex spectral analysis of autofluorescent profiles. Additionally, we are working on using optical signatures such as these to facilitate physical isolation of epidermal cell populations using flow cytometry-based strategies such as fluorescent activated cell sorting (FACS) for the purposes of generating single source genetic profiles from touch mixtures. Although previous work suggests that analyzing DNA profiles directly from isolated epidermal cells may be a challenge due to the prevalence of extracellular or ‘cell-free’ DNA in touch samples16, the sheer quantity of cells that may be recovered from these sample types (up to ~1×105,16) may help to overcome such obstacles.\n\n\nData availability\n\nF1000Research: Dataset 1. Flow cytometry source data for individual contributors, 10.5256/f1000research.8036.d11374917", "appendix": "Author contributions\n\n\n\nCE conceived the study. CE, CS, KP, EB, and YK designed the experiments. CS, EB, YK carried out the research. KP assisted with data analysis and provided expertise in the area of forensic casework. CE, CS, and KP 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 project was funded by the National Institute of Justice Award number 2013-DN-BX-K033 (PI: Ehrhardt). Flow cytometry services in support of the project were provided by the VCU Massey Cancer Center, supported in part with funding from NIH-NCI P30CA016059.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThe authors gratefully acknowledge Daniel Conrad and Julie Farnsworth for providing technical assistance for this project.\n\n\nReferences\n\nBudowle B, Onorato AJ, Callaghan TF, et al.: Mixture interpretation: defining the relevant features for guidelines for the assessment of mixed DNA profiles in forensic casework. J Forensic Sci. 2009; 54(4): 810–821. PubMed Abstract \| Publisher Full Text\n\nDean L, Kwon YJ, Philpott MK, et al.: Separation of uncompromised whole blood mixtures for single source STR profiling using fluorescently-labeled human leukocyte antigen (HLA) probes and fluorescence activated cell sorting (FACS). Forensic Sci Int Genet. 2015; 17: 8–16. PubMed Abstract \| Publisher Full Text\n\nVerdon TJ, Mitchell RJ, Chen W, et al.: FACS separation of non-compromised forensically relevant biological mixtures. Forensic Sci Int Genet. 2015; 14: 194–200. PubMed Abstract \| Publisher Full Text\n\nAnslinger K, Bayer B, Mack B, et al.: Sex-specific fluorescent labelling of cells for laser microdissection and DNA profiling. Int J Legal Med. 2007; 121(1): 54–56. PubMed Abstract \| Publisher Full Text\n\nGielen V, Schmitt D, Thivolet J: HLA class I antigen (heavy and light chain) expression by Langerhans cells and keratinocytes of the normal human epidermis: ultrastructural quantitation using immunogold labelling procedure. Arch Dermatol Res. 1988; 280(3): 131–136. PubMed Abstract \| Publisher Full Text\n\nHaftek M, Viac J, Cordier G, et al.: Flow cytometry for separation of keratinocyte subpopulations from the viable epidermis. J Invest Dermatol. 1986; 87(4): 480–484. PubMed Abstract\n\nBrancaleon L, Lin G, Kollias N: The in vivo fluorescence of tryptophan moieties in human skin increases with UV exposure and is a marker for epidermal proliferation. J Invest Dermatol. 1999; 113(6): 977–982. PubMed Abstract \| Publisher Full Text\n\nSylvestre JP, Bouissou CC, Guy RH, et al.: Extraction and quantification of amino acids in human stratum corneum in vivo. Br J Dermatol. 2010; 163(3): 458–465. PubMed Abstract \| Publisher Full Text\n\nFereidouni F, Bader AN, Colonna A, et al.: Phasor analysis of multiphoton spectral images distinguishes autofluorescence components of in vivo human skin. J Biophotonics. 2014; 7(8): 589–596. PubMed Abstract \| Publisher Full Text\n\nGildenast T, Lasch J: Isolation of ceramide fractions from human stratum corneum lipid extracts by high-performance liquid chromatography. Biochim Biophys Acta. 1997; 1346(1): 69–74. PubMed Abstract \| Publisher Full Text\n\nBissonnette R, Zeng H, McLean DI, et al.: Psoriatic plaques exhibit red autofluorescence that is due to protoporphyrin IX. J Invest Dermatol. 1998; 111(4): 586–591. PubMed Abstract \| Publisher Full Text\n\nGillies R, Zonios G, Anderson RR, et al.: Fluorescence excitation spectroscopy provides information about human skin in vivo. J Invest Dermatol. 2000; 115(4): 704–707. PubMed Abstract \| Publisher Full Text\n\nKjeldstad B, Johnsson A, Sandberg S: Influence of pH on porphyrin production in Propionibacterium acnes. Arch Dermatol Res. 1984; 276(6): 396–400. PubMed Abstract \| Publisher Full Text\n\nPiruska A, Nikcevic I, Lee SH, et al.: The autofluorescence of plastic materials and chips measured under laser irradiation. Lab Chip. 2005; 5(12): 1348–1354. PubMed Abstract \| Publisher Full Text\n\nMeyer S, Cartelat A, Moya I, et al.: UV-induced blue-green and far-red fluorescence along wheat leaves: a potential signature of leaf ageing. J Exp Bot. 2003; 54(383): 757–769. PubMed Abstract \| Publisher Full Text\n\nStanciu CE, Philpott MK, Kwon YJ, et al.: Optical characterization of epidermal cells and their relationship to DNA recovery from touch samples [version 1; referees: 2 approved]. F1000Res. 2015; 4: 1360. Publisher Full Text\n\nStanciu CE, Philpott MK, Bustamante EE, et al.: Dataset 1 in: Analysis of red autofluorescence (650–670nm) in epidermal cell populations and its potential for distinguishing contributors to 'touch' biological samples. F1000Research. 2016. Data Source" }	[ { "id": "16701", "date": "28 Oct 2016", "name": "Timothy J Verdon", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 skin cell analysis by FACS in forensic biological analysis, and present some results which shed some light on the potential, or lack thereof, for this kind of technique. The experiment is technically sound and well presented, and although there is no statistical weight placed on the differences or similarities among cell species, some trends are obvious.\nSome minor amendments and areas for future examination would improve the paper:\nIt must be noted earlier on (i.e. at the end of the first introductory paragraph) that much of the DNA in touch samples will be exogenous - the use of cell sorting for these kinds of samples may not actually yield appropriate quantities of DNA for profiling.\n\nThe methodology of collecting touch samples raises some questions - the swab type used may have an effect on the cell structure and elution of the sample, possibly even may affect / introduce autofluorescence in some cases. Also were the swabs dried post-sampling or eluted straight afterwards? In actual casework this may also affect the quality of the keratinocytes, as will the lag between sampling and analysis. It would be of benefit to repeat this study with older samples, perhaps placed onto a swab and dried for a week before analysis, compared to pristine cells collected via a glass bead method1 without the need for swabs at all.\n\nThe cells collected and analysed herein may not all be borne of the individual who touched the item - how controlled were the volunteers before touching? Did they have to wash their hands? Did they have contact with others or other work environments? The only way to assess this fully would be to examine the profiles from these samples post-analysis.\n\nFollowing this, the samples may not actually yield sufficient DNA, as rightly pointed out in the discussion. It seems a shame that these samples were not separated and profiled to examine this further.\n\nOverall, the publication of this type of data should be encouraged, and hopefully more thorough experiments of this kind will follow on from this work.", "responses": [] }, { "id": "18017", "date": "28 Nov 2016", "name": "Mark Perlin", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article’s title and abstract are appropriate. The design, methods and analysis have been explained, and are appropriate for this preliminary report. The conclusions are supported by the results. Data have been provided. Some context and suggestions for analysis are provided here.\n\nSeparation of cellular genetic material is central to forensic DNA analysis. Separation stages include:\nCells. Labs separate cells out of mixed cellular populations based on physical characteristics. For example, differential extraction can enrich sperm in one cell fraction relative to epithelial cells in another fraction. The red autofluorescence described in the paper occurs at this front-end stage.\n\nDNA. Extraction procedures separate nucleic acids from other molecules. Such procedures include organic, non-organic, chelex, FTA or silica methods.\n\nLoci. PCR separates short DNA regions from the rest of the genome by amplifying (i.e., purifying) these specific regions relative to background sequence.\n\nFragments. Fluorescent electrophoresis separates DNA fragments by size, identifying length polymorphism alleles and determining their relative quantity.\n\nGenotype. Computer separation of electrophoretic data can determine the genotypes of each contributor to a DNA mixture1. Back-end methods that do not separate contributors2 have limited applicability for resolving complex DNA mixtures.\n\nThis paper focuses on the first step, front-end separation of mixed epithelial cell populations. The authors investigate a novel physical assay – red autofluorescence in the 650-670 nm range. Their preliminary results demonstrate effective separation, with some cell populations clearly distinguished from others based on fluorescent frequency.\n\nThere are some limitations to the current procedure. The partial autofluorescence separation is incomplete, and can vary for the same subject between samplings. However, as the authors note, “the ability to separate out even one contributor [subset] may render the remaining mixture more interpretable in downstream DNA analysis.” This ability, coupled with automatic fluorescent activated cell sorting (FACS) and automatic DNA mixture data analysis (e.g., TrueAllele® computing), could be quite powerful.\n\nJoint Bayesian analysis of multiple STR samplings of low-level DNA mixtures can recover considerable identification information3. The autofluorescence method reported here could produce analogous data by taking FACS samplings at different fluorescent frequencies. Joint computer analysis of these different frequency-sampled STR amplifications would then complete the (mathematical) mixture separation into informative (probabilistic) genotypes.\n\nThe authors propose a novel front-end DNA separation method. As with most front-end methods, the cell separation is incomplete. However, their autofluorescence separation can become more complete if used in conjunction with other front-end discrimination methods. Moreover, combining their front-end cell separation with available back-end computer data separation1,3 could extract considerable identification information from complex DNA mixtures.", "responses": [] } ]	1	https://f1000research.com/articles/5-180
https://f1000research.com/articles/5-179/v1	16 Feb 16	{ "type": "Review", "title": "Contemporary Management of Prostate Cancer", "authors": [ "Katherine Cotter", "Badrinath Konety", "Maria A. Ordonez", "Badrinath Konety", "Maria A. Ordonez" ], "abstract": "Prostate cancer represents a spectrum ranging from low-grade, localized tumors to devastating metastatic disease. We discuss the general options for treatment and recent developments in the field.", "keywords": [ "prostate", "cancer", "tumor" ], "content": "\n\nProstate cancer is the most common non-cutaneous malignancy in American men and the second most common cause of cancer-specific mortality. In 2015, prostate cancer will be diagnosed in an estimated 220,800 men in the United States, and an estimated 27,540 men will die of the disease (http://www.cancer.gov/cancertopics/types/prostate)1. Determining which of these cancers are likely to progress remains a significant challenge in management. Novel translational research has helped to guide these decisions to maximize oncologic outcomes while minimizing morbidity of overtreatment. Integration of tumor biology with clinical practice may lead to a more individualized, patient-specific treatment plan.\n\nAdditionally, novel treatment options aim to achieve a higher therapeutic index. This article reviews the general management of both localized and metastatic prostate cancer, with a focus on emerging research aimed at guiding both management decisions and developments in targeted therapies.\n\nRisk stratification of clinically localized prostate cancer has served as a guide to counsel patients on treatment options (Table 1)2,3. The American Urological Association (AUA) guidelines offer active surveillance (AS), radical prostatectomy (RP) with or without pelvic lymphadenectomy, external beam radiotherapy (EBRT), and interstitial radiotherapy/brachytherapy for clinically localized disease (T1, T2), with an impetus toward treatment with higher risk tumors4. Limited evidence has led to a lack of consensus regarding the preferred treatment.\n\nAdapted from 2.\n\n\nProstate cancer detection\n\nDue to a shift in disease stage at presentation, the proportion of patients presenting with high-risk or metastatic disease has declined, as have the death rates. In 1990, the 5-year relative survival rate of prostate cancer was 88.4%, while in 2007, 5-year survival was 99.7% (Surveillance, Epidemiology, and End Results [SEER] Medicare Cancer Statistics, 2007–2011); this demonstrates a potential benefit from early detection. However, aggressive screening and superior modes of detection carry the risk of overtreatment. Analysis of greater than 10,000 men in the Cancer of the Prostate Strategic Urologic Research Endeavor (CaPSURE) registry in 2007 showed a significant increase in clinical T1c disease and a transition toward greater use of AS. The CaPSURE registry consists of patients from a total of 31 U.S. centers with biopsy-proven prostate adenocarcinoma. From 2000–2006, just over 50% of all new prostate cancer diagnoses within the registry were low risk (prostate-specific antigen [PSA] <10 ng/mL, Gleason score ≤6, and clinical stage ≤Ta). Within this group of patients, parameters such as percentage of biopsy cores positive and stratification of the individual Gleason scores5 have subdivided this population further. This study demonstrated a significant shift toward low-risk characteristics and a potential under-use of AS as an option. Therefore, use of the Cancer of the Prostate Risk Assessment (CAPRA) score, a risk stratification tool, has allowed substratification even within a low-risk population to predict biochemical recurrence and counsel patients more effectively6.\n\nAdvances in imaging have also improved the detection of prostate cancer. Multi-parametric magnetic resonance imaging (mp-MRI)7 incorporates functional parameters to T2-weighted imaging, providing dynamic imaging of prostate lesions. A recently published prospective cohort study compared standard 12-core ultrasound-guided biopsy with targeted, MRI-guided fusion biopsy in over 1000 men with at least one prior negative biopsy from 2007–2014. Each patient had an mp-MRI, and those with suspicious lesions underwent fusion biopsy, followed by a standard ultrasound-guided biopsy by another urologist unaware of the MRI results. Results showed that fusion biopsy was able to detect high-grade prostate cancer with higher sensitivity than standard biopsy alone (77% versus 53%, respectively); however, fusion biopsy demonstrated lower sensitivity in detecting low-grade disease. The long-term clinical significance remains to be shown but points to improved detection of clinically significant prostate cancer8.\n\nIn an effort to further risk stratify prostate cancer patients and find markers for aggressive disease, genomic biomarkers have been integrated into clinical practice and include Prolaris (Myriad Genetics), Oncotype DX Prostate Cancer Assay (Genomics Health, Inc), transmembrane protease, serine 2 (TMPRSS-2), and prostate cancer antigen 3 (PCA3)7. Oncotype DX tests for specific gene expression in prostate biopsy tissue and, in conjunction with National Comprehensive Cancer Network (NCCN) risk criteria, can be used to determine candidacy for AS9. The Prolaris test assesses the expression of genes primarily involved in cell cycle progression to directly evaluate tumor growth and determine the likelihood of disease progression10. While further studies are warranted, such biomarkers have been a promising area of investigation.\n\nRecent research has investigated the application of kallikrein-based tests to supplement PSA testing. Integration of kallikrein markers, which include free PSA (fPSA), single-chain intact PSA (iPSA), total PSA (tPSA), and human kallikrein 2 (hK2), has shown increased specificity in predictive models at PSA 2–10 ng/mL. The potential value of these markers would be of greatest use for patients with PSA of 2–10 ng/ml and low PSA density based on digital rectal examination11.\n\nIn addition, proPSA, a molecular, inactive precursor of PSA, has also been posited as a potential marker, specifically the truncated (-2) form. hK2, a kallikrein-related peptidase, cleaves proPSA into the active form. Patients with prostate cancer may demonstrate elevated levels of proPSA than patients without cancer12. The prostate health index (PHI) combines tPSA, % fPSA, and (-2) proPSA. Further areas of investigation include prospective studies and applicability in patients with a strong family history of prostate cancer as well as those on 5-α-reductase inhibitors13.\n\nNewer diagnostic imaging modalities used mainly to detect disease recurrence after definitive therapy include prostate-specific membrane antigen based positron emission tomography (PSMA PET), C11 choline PET, and sodium fluoride bone scan. A recent prospective analysis of 38 men with biochemical recurrence (mean PSA 1.74 ng/mL) following either RP or EBRT showed that PSMA PET demonstrated greater sensitivity than a standard (18)F-fluoromethylcholine PET to detect disease14. As the PSA window for salvage therapy is often lower than the threshold reliably detected by standard PET, such imaging options may allow patients to have treatment at an earlier stage. Retrospective analysis of C11 PET has been predictive of positive findings for recurrence with a PSA of 1.24 ng/mL or PSA velocity of 1.32 ng/mL/year15. Both fluorocholine and sodium fluoride PET computed tomography (CT) bone scans showed ability to detect bony metastases specifically, when studied in a prospective series of 42 prostate cancer patients with a minimum 6-month follow-up period16.\n\n\nTreatment\n\nAccording to the AUA guidelines regarding the management of clinically localized prostate cancer, each patient should be informed about the risks and benefits of available initial interventions, including AS, EBRT, brachytherapy, and RP3.\n\nOne randomized clinical trial showed a reduction in overall mortality with RP versus watchful waiting17, as well as a reduction in disease-specific death, local progression, and metastasis. The Prostate Cancer Intervention versus Observation Trial (PIVOT) trial, which compared RP to AS, enrolled a more contemporary, screen-detected cohort of men and showed a significant benefit in overall survival with RP only in patients with pre-treatment PSA >10 ng/ml or high-risk disease18, but no significant difference in patients with low-risk cancer. These findings support the role of AS as an option in patients with low-volume, low-grade disease. However, the question frequently remains which patients best qualify for AS. Novel tools, such as prostate MRI and serum and urinary biomarkers, aim to provide a more accurate method of determining which patients are appropriate for AS19. Cost may prove a limiting factor in the widespread use of these tools but, when used in conjunction with PSA, Gleason score, and clinical stage, may translate to more informed patient decision making.\n\n\nRadiation therapy\n\nEBRT and brachytherapy may be offered as monotherapy or in conjunction depending on patient and tumor factors; hormone therapy may also play a synergistic role in patients with intermediate- and high-risk disease by promoting cellular apoptosis3. A 6-month course of neoadjuvant hormonal therapy has demonstrated a survival benefit in intermediate-risk patients ultimately receiving EBRT, and adjuvant hormonal therapy has been shown to prolong survival in high-risk patients and/or patients with locally advanced disease20,21, particularly with a 3-year course of hormonal therapy after EBRT22,23. Hence, combined androgen deprivation therapy (ADT) and EBRT should be discussed and offered to patients with locally advanced prostate cancer. Two randomized controlled clinical trials demonstrated that high-dose radiation may reduce the risk of PSA recurrence24. Methods to target tumors with increasing accuracy, including the advent of intensity-modulated radiotherapy, have led to greater dose escalation of radiation with a wider margin of safety25.\n\nPrimary hormonal monotherapy may be offered in the setting of limited life expectancy and in patients unable to proceed with other local therapies26. Benefits of treatment, however, must be weighed against potential complications impacting the patient’s quality of life, including hot flashes, truncal obesity, increased risk of metabolic syndrome, and cardiovascular side effects (particularly in men with pre-existing cardiac disease). Additional treatment options for localized prostate cancer include cryotherapy and high-intensity focused ultrasound (HIFU), neither of which are currently recommended first-line treatments in the United States.\n\n\nAlternate therapies\n\nIn addition to the standard treatment options outlined above, prostate cryotherapy and HIFU are alternate therapies that are less widely available for the treatment of localized prostate cancer. A review of cryotherapy outcomes showed 5-year biochemical survival rates of approximately 70% for patients undergoing whole gland ablation. Patients in this group were also found to have higher rates of erectile dysfunction27. Similarly, HIFU therapy for prostate cancer demonstrated 5- and 10-year biochemical survival rates of 80% and 61%. Potency was preserved in 25% of patients after treatment. Improvements in imaging, such as mp-MRI, aim to optimize the therapeutic index of focal therapy. This burgeoning field couples mp-MRI with ablative techniques to treat index prostate lesions. Furthermore, mp-MRI has allowed for more accurate follow-up after definitive focal therapy. Cryotherapy and brachytherapy, as well as HIFU (in Europe), are established focal therapy modalities; however, newer techniques, such as irreversible electroporation (Nanoknife™)28 and laser interstitial therapy, are under current investigation and may demonstrate promise.\n\n\nDisease recurrence after local therapy\n\nIn the majority of patients, RP is curative; however, data suggests that within 10 years after surgery, up to 33% of men will have evidence of recurrent disease29. The risk is greater in patients with adverse surgical pathologic features, including extraprostatic extension, positive surgical margins, and seminal vesicle invasion30. Adjuvant radiation may be offered to these patients and has been shown to reduce the risk of local and PSA recurrence as well as clinical progression31.\n\nBiochemical recurrence after prostatectomy is defined as a PSA of ≥0.2 ng/mL on two separate tests. Data indicates that the salvage radiotherapy is most effective when administered with a low PSA, i.e. 0.5 to 1.5 ng/mL32. Options include surveillance, salvage radiation, ADT, and enrolment in clinical trials. In the post-radiation setting, biochemical failure is defined as PSA ≥2.0 ng/mL over the nadir, or three consecutive rises33. Options include surveillance, salvage prostatectomy, ADT, cryotherapy, and clinical trials.\n\nPre-treatment nomograms can be used to determine an individual patient’s risk of specific clinical endpoints and therefore may supplement the patient’s discussion about the need for multimodal therapy. Specifically, pre-prostatectomy nomograms predict the risk of adverse pathologic features, as well as of disease recurrence. Models, such as the Kattan nomogram and University of California, San Francisco (UCSF) CAPRA score34, use pre-treatment PSA, biopsy results, clinical stage, and other factors to estimate certain outcomes.\n\nThe D’Amico classification uses PSA, Gleason score, and clinical stage to risk stratify patients into low-, intermediate-, and high-risk categories2. Other models, such as the UCSF-CAPRA score, stratify estimated risk using a 0–10 numeric scale calculated with patient information including age and PSA at diagnosis, Gleason score, clinical stage, and percent of biopsy cores involved34. Nomograms can provide patients and physicians with objective information to select treatment plans and estimate risk.\n\nWhen biochemical recurrence has occurred, PSA kinetics can be used to calculate the risk of local versus distant recurrence, as well as to guide indications for obtaining bone scans and other imaging studies. Patients with a PSA doubling time (PSADT) of >15 months have a low cancer-specific mortality rate at 10 years and therefore may be candidates for AS, particularly if life expectancy is <10 years35. Conversely, a PSADT of 3 months or under suggests distant metastatic disease and a median 6-year survival.\n\n\nAdvanced disease\n\nLocally advanced and distant metastatic disease frequently require a multimodal treatment approach. For locally advanced prostate cancer, main treatment options include EBRT with interstitial radiotherapy, RP with hormonal therapy, and EBRT with hormonal therapy, as discussed above. In the setting of PSA rise post-treatment, time to PSA recurrence, PSADT, and Gleason score may be predictive of progression to metastatic disease36. Therefore, these parameters may be used to determine which therapies are best suited for the patient given the likelihood of disease progression.\n\nOptions for advanced systemic prostate cancer with the aim to achieve castrate-levels of testosterone include bilateral orchiectomy (surgical castration), luteinizing hormone receptor analogs with or without complete androgen blockade, androgen receptor (AR) antagonists (steroidal or non-steroidal), and ketoconazole with steroids. Future directions in the field of prostate cancer management include RP for advanced and oligometastatic disease in the context of combined modality therapy. Patients with metastatic castration-resistant prostate cancer (mCRPC) may be candidates for chemotherapy or immunotherapy depending on prior therapies received, presence and severity of symptoms, documented metastases on imaging, and performance status3. Sipuleucel-T, an autologous cellular immunotherapy, is an option for men with good performance status, no prior docetaxel therapy or visceral metastases37, and symptoms from metastases not requiring narcotic medication. The Immunotherapy for Prostate Adenocarcinoma Treatment (IMPACT) trial demonstrated a 4.1-month survival advantage with sipuleucel-T versus placebo, although no effect on time to disease progression was found38. Cabazitaxel, a tubulin-binding taxane, may be offered in the post-docetaxel setting and was shown to have greater overall (15.1 versus 12.7 months, p <0.0001) and progression-free survival (2.8 versus 1.4 months, p <0.0001) when compared to mitoxantrone39. Abiraterone is an androgen biosynthesis inhibitor shown to prolong survival in men with mCRPC after receiving chemotherapy. Enzalutamide, a targeted AR inhibitor, has been shown to improve disease-free and overall survival rates in men with mCRPC who had previously received chemotherapy40. It has also been shown to extend time to radiographic progression and death as well as improve overall survival in men with mCRPC prior to receiving chemotherapy. Delay in time to chemotherapy was also reported41. Similar to enzalutamide, abiraterone was also shown to delay radiographic progression and time to chemotherapy in men with mCRPC42.\n\nIn the setting of symptomatic bony metastases, radium-223 can be used. Recent data suggests that selecting the optimum combination and/or sequence of treatments may play a significant role in future responsiveness to therapies, particularly those with similar mechanisms of action43.\n\nRecent studies demonstrate that multiple factors contribute to AR reactivation and CRPC, despite castrate serum levels of androgens. Various mechanisms include changes in AR expression, structural modification through gene amplification, mutation, and alternative splicing44. Therefore, agents that work via the CYP17 pathway may be required in mCRPC.\n\nNovel agents with activity on the CYP17 pathway, such as galeterone, or modulators of AR signaling provide an alternative to abiraterone and enzalutamide in the setting of castration resistance45. Ongoing research continues in the potential synergistic relationship between CYP17 inhibitors and antiandrogens46. Biomarkers predictive of response or resistance may promote the best use of these treatments in the future.\n\nPatients receiving ADT are at a greater risk for osteoporosis and bone-related complications that can significantly increase morbidity. Men with mCRPC are at an even greater risk for osteoporosis and skeletal-related events (SREs)47. Prospective studies of men receiving ADT demonstrate a decrease in bone mineral density of 3% at the lumbar spine (1.4% to 3.3%) and 2% at the hip (0.7% to 3.3%) within the first year of treatment48. Options for treatment-related osteoporosis thereafter include bisphosphonates, denosumab (which is a receptor activator of nuclear factor-kappaB [RANK] ligand inhibitor), and selective estrogen receptor modulators.\n\nZoledronic acid (Zometa), a bisphosphonate, inactivates osteoclastic activity. When compared to placebo in the setting of bony metastases and mCRPC, zoledronic acid was associated with fewer SREs at 15 months when compared with placebo (33.2% versus 44.2%; p = 0.021). Time to first SRE was improved with zoledronic acid (488 versus 321 days; p = 0.009)49. Current evidence supports the monthly use of either zoledronic acid or denosumab for the reduction of SREs in men with bone-metastatic castration-resistant disease.\n\nDenosumab is a human monoclonal antibody against nuclear factor-kappaB ligand (RANK ligand) involved in bone turnover, thus inhibiting osteoclast activity and subsequent bone breakdown. This medication was approved in 2010 for the prevention of SREs in patients with mCRPC. A randomized controlled trial demonstrated that denosumab was superior to Zometa in preventing SREs in mCRPC50. The AUA guidelines recommend either medication for patients with bony metastases and castration resistance. Due to the risk of hypocalcemia with both agents, vitamin D, calcium, and frequent serum calcium monitoring is critical. Given the risk of renal insufficiency with the use of zoledronic acid, denosumab may be the preferred agent in patients with chronic kidney disease.\n\n\nAreas of future research/future directions in the field\n\nFuture directions in the field of prostate cancer management include RP for advanced and oligometastatic disease in the context of combined modality therapy51 and the role of new immunotherapeutic agents, such as programmed cell death protein (PD-1) and PD-L1 inhibitors52,53. For localized disease, greater use of focal therapies such as HIFU, vapor therapy such as REZUM, and laser ablation are all areas of future research.\n\n\nConclusion\n\nA shift toward understanding individual tumor behavior and clinical prognostic information provides a more tailored treatment plan for patients with prostate cancer. Future directions for research include precision medicine with individualized genetic analysis and targeted therapy. These concepts represent areas of further investigation. Advances in currently available treatments translate to a wider therapeutic window, which can maximize patient benefit while minimizing morbidity.\n\n\nAbbreviations\n\nADT: androgen deprivation therapy\n\nAR: androgen receptor\n\nAS: active surveillance\n\nAUA: American Urological Association\n\nCaPSURE registry: Cancer of the Prostate Strategic Urologic Research Endeavor\n\nCAPRA: Cancer of the Prostate Risk Assessment\n\nCT: computed tomography\n\nEBRT: external beam radiotherapy\n\nfPSA: free PSA\n\nHIFU: high-intensity focused ultrasound\n\nhK2: human kallikrein 2, a prostate-specific kallikrein (protease) produced by prostate epithelium\n\nIMPACT: Immunotherapy for Prostate Adenocarcinoma Treatment\n\niPSA: intact prostate-specific antigen\n\nmCRPC: metastatic castration-resistant prostate cancer\n\nMRI: magnetic resonance imaging\n\nNCCN: National Comprehensive Cancer Network\n\nPCA3: prostate cancer antigen 3\n\nPD-1: programmed cell death protein\n\nPET: positron emission tomography\n\nPHI: prostate health index\n\nPIVOT: Prostate Cancer Intervention versus Observation Trial\n\nPSA: prostate-specific antigen\n\nPSADT: prostate-specific antigen doubling time\n\nPSMA: prostate-specific membrane antigen\n\nRANK: receptor activator of nuclear factor-kappaB\n\nRP: radical prostatectomy\n\nSEER: Surveillance, Epidemiology, and End Results\n\nSRE: skeletal-related events\n\nT1c: clinical stage whereby prostate cancer is detected based on PSA value\n\nTMPRSS-2: transmembrane protease, serine 2\n\ntPSA: total prostate-specific antigen", "appendix": "Competing interests\n\n\n\nThe author(s) declare that they 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\nProstate Cancer. National Cancer Institute, 2015. Reference Source\n\nD'Amico AV, Whittington R, Malkowicz SB, et al.: Biochemical outcome after radical prostatectomy, external beam radiation therapy, or interstitial radiation therapy for clinically localized prostate cancer. JAMA. 1998; 280(11): 969–974. PubMed Abstract \| Publisher Full Text\n\nYarbro JW, Page DL, Fielding LP, et al.: American Joint Committee on Cancer prognostic factors consensus conference. Cancer. 1999; 86(11): 2436–2446. PubMed Abstract \| Publisher Full Text\n\nThompson I, Thrasher JB, Aus G, et al.: Guideline for the management of clinically localized prostate cancer. American Urological Association Guidelines. Accessed August 1, 2015. Reference Source\n\nBostwick DG: Grading prostate cancer. Am J Clin Pathol. 1994; 102(4 Suppl 1): S38–56. PubMed Abstract\n\nCooperberg MR, Broering JM, Kantoff PW, et al.: Contemporary trends in low risk prostate cancer: risk assessment and treatment. J Urol. 2007; 178(3 Pt 2): S14–9. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nRaskolnikov D, George AK, Rais-Bahrami S, et al.: The Role of Magnetic Resonance Image Guided Prostate Biopsy in Stratifying Men for Risk of Extracapsular Extension at Radical Prostatectomy. J Urol. 2015; 194(1): 105–11. PubMed Abstract \| Publisher Full Text\n\nSiddiqui MM, Rais-Bahrami S, Turkbey B, et al.: Comparison of MR/ultrasound fusion-guided biopsy with ultrasound-guided biopsy for the diagnosis of prostate cancer. JAMA. 2015; 313(4): 390–397. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nBratt O, Lilja H: Serum markers in prostate cancer detection. Curr Opin Urol. 2015; 25(1): 59–64. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKnezevic D, Goddard AD, Natraj N, et al.: Analytical validation of the Oncotype DX prostate cancer assay - a clinical RT-PCR assay optimized for prostate needle biopsies. BMC Genomics. 2013; 14: 690. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nCooperberg MR, Simko JP, Cowan JE, et al.: Validation of a cell-cycle progression gene panel to improve risk stratification in a contemporary prostatectomy cohort. J Clin Oncol. 2013; 31(11): 1428–1434. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nVickers AJ, Cronin AM, Roobol MJ, et al.: A four-kallikrein panel predicts prostate cancer in men with recent screening: data from the European Randomized Study of Screening for Prostate Cancer, Rotterdam. Clin Cancer Res. 2010; 16(12): 3232–3239. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVickers A, Cronin A, Roobol M, et al.: Reducing unnecessary biopsy during prostate cancer screening using a four-kallikrein panel: an independent replication. J Clin Oncol. 2010; 28(15): 2493–2498. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nLoeb S, Catalona WJ: The Prostate Health Index: a new test for the detection of prostate cancer. Ther Adv Urol. 2014; 6(2): 74–77. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMorigi JJ, Stricker PD, van Leeuwen PJ, et al.: Prospective Comparison of 18F-Fluoromethylcholine Versus 68Ga-PSMA PET/CT in Prostate Cancer Patients Who Have Rising PSA After Curative Treatment and Are Being Considered for Targeted Therapy. J Nucl Med. 2015; 56(8): 1185–1190. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nDusing RW, Peng W, Lai SM, et al.: Prostate-specific antigen and prostate-specific antigen velocity as threshold indicators in 11C-acetate PET/CTAC scanning for prostate cancer recurrence. Clin Nucl Med. 2014; 39(9): 777–783. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nBill-Axelson A, Holmberg L, Ruutu M, et al.: Radical prostatectomy versus watchful waiting in early prostate cancer. N Engl J Med. 2005; 352(19): 1977–1984. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nWilt TJ, Brawer MK, Jones KM, et al.: Radical prostatectomy versus observation for localized prostate cancer. N Engl J Med. 2012; 367(3): 203–213. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nvan den Bergh RC, Ahmed HU, Bangma CH, et al.: Novel tools to improve patient selection and monitoring on active surveillance for low-risk prostate cancer: a systematic review. Eur Urol. 2014; 65(6): 1023–1031. PubMed Abstract \| Publisher Full Text\n\nBolla M, Collette L, Blank L, et al.: Long-term results with immediate androgen suppression and external irradiation in patients with locally advanced prostate cancer (an EORTC study): a phase III randomised trial. Lancet. 2002; 360(9327): 103–106. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nD'Amico AV, Manola J, Loffredo M, et al.: 6-month androgen suppression plus radiation therapy vs radiation therapy alone for patients with clinically localized prostate cancer: a randomized controlled trial. JAMA. 2004; 292(7): 821–827. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nBolla M, de Reijke TM, Van Tienhoven G, et al.: Duration of androgen suppression in the treatment of prostate cancer. N Engl J Med. 2009; 360(24): 2516–2527. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nWarde P, Mason M, Ding K, et al.: Combined androgen deprivation therapy and radiation therapy for locally advanced prostate cancer: a randomised, phase 3 trial. Lancet. 2011; 378(9809): 2104–2111. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nZietman AL, DeSilvio ML, Slater JD, et al.: Comparison of conventional-dose vs high-dose conformal radiation therapy in clinically localized adenocarcinoma of the prostate: a randomized controlled trial. JAMA. 2005; 294(10): 1233–1239. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nBauman G, Rumble RB, Chen J, et al.: Intensity-modulated radiotherapy in the treatment of prostate cancer. Clin Oncol (R Coll Radiol). 2012; 24(7): 461–473. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nMeng MV, Grossfeld GD, Sadetsky N, et al.: Contemporary patterns of androgen deprivation therapy use for newly diagnosed prostate cancer. Urology. 2002; 60(3 Suppl 1): 7–11, discussion 11–2. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nJones JS, Rewcastle JC, Donnelly BJ, et al.: Whole gland primary prostate cryoablation: initial results from the cryo on-line data registry. J Urol. 2008; 180(2): 554–558. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nValerio M, Dickinson L, Ali A, et al.: A prospective development study investigating focal irreversible electroporation in men with localised prostate cancer: Nanoknife Electroporation Ablation Trial (NEAT). Contemp Clin Trials. 2014; 39(1): 57–65. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAmling CL, Blute ML, Bergstralh EJ, et al.: Long-term hazard of progression after radical prostatectomy for clinically localized prostate cancer: continued risk of biochemical failure after 5 years. J Urol. 2000; 164(1): 101–105. PubMed Abstract \| Publisher Full Text\n\nStephenson AJ, Scardino PT, Eastham JA, et al.: Preoperative nomogram predicting the 10-year probability of prostate cancer recurrence after radical prostatectomy. J Natl Cancer Inst. 2006; 98(10): 715–717. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThompson IM Jr, Tangen CM, Paradelo J, et al.: Adjuvant radiotherapy for pathologically advanced prostate cancer: a randomized clinical trial. JAMA. 2006; 296(19): 2329–2335. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nSwanson GP, Hussey MA, Tangen CM, et al.: Predominant treatment failure in postprostatectomy patients is local: analysis of patterns of treatment failure in SWOG 8794. J Clin Oncol. 2007; 25(16): 2225–2229. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nConsensus statement: guidelines for PSA following radiation therapy. American Society for Therapeutic Radiology and Oncology Consensus Panel. Int J Radiat Oncol Biol Phys. 1997; 37(5): 1035–1041. PubMed Abstract \| Publisher Full Text\n\nCooperberg MR, Pasta DJ, Elkin EP, et al.: The University of California, San Francisco Cancer of the Prostate Risk Assessment score: a straightforward and reliable preoperative predictor of disease recurrence after radical prostatectomy. J Urol. 2005; 173(6): 1938–1942. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nFreedland SJ, Humphreys EB, Mangold LA, et al.: Risk of prostate cancer-specific mortality following biochemical recurrence after radical prostatectomy. JAMA. 2005; 294(4): 433–439. PubMed Abstract \| Publisher Full Text\n\nFreedland SJ, Humphreys EB, Mangold LA, et al.: Death in patients with recurrent prostate cancer after radical prostatectomy: prostate-specific antigen doubling time subgroups and their associated contributions to all-cause mortality. J Clin Oncol. 2007; 25(13): 1765–1771. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nDi Lorenzo G, Ferro M, Buonerba C: Sipuleucel-T (Provenge®) for castration-resistant prostate cancer. BJU Int. 2012; 110(2 Pt 2): E99–104. PubMed Abstract \| Publisher Full Text\n\nKantoff PW, Higano CS, Shore ND, et al.: Sipuleucel-T immunotherapy for castration-resistant prostate cancer. N Engl J Med. 2010; 363(5): 411–422. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nde Bono JS, Oudard S, Ozguroglu M, et al.: Prednisone plus cabazitaxel or mitoxantrone for metastatic castration-resistant prostate cancer progressing after docetaxel treatment: a randomised open-label trial. Lancet. 2010; 376(9747): 1147–1154. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nScher HI, Fizazi K, Saad F, et al.: Increased survival with enzalutamide in prostate cancer after chemotherapy. N Engl J Med. 2012; 367(13): 1187–1197. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nBeer TM, Armstrong AJ, Rathkopf DE, et al.: Enzalutamide in metastatic prostate cancer before chemotherapy. N Engl J Med. 2014; 371(5): 424–433. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nRyan CJ, Smith MR, de Bono JS, et al.: Abiraterone in metastatic prostate cancer without previous chemotherapy. N Engl J Med. 2013; 368(2): 138–148. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nCrawford ED, Higano CS, Shore ND, et al.: Treating Patients with Metastatic Castration Resistant Prostate Cancer: A Comprehensive Review of Available Therapies. J Urol. 2015; 194(6): 1537–1547. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nBambury RM, Rathkopf DE: Novel and next-generation androgen receptor-directed therapies for prostate cancer: Beyond abiraterone and enzalutamide. Urol Oncol. 2015; pii: S1078-1439(15)00269-0. PubMed Abstract \| Publisher Full Text\n\nShafi AA, Yen AE, Weigel NL: Androgen receptors in hormone-dependent and castration-resistant prostate cancer. Pharmacol Ther. 2013; 140(3): 223–238. PubMed Abstract \| Publisher Full Text\n\nPinto Á: Beyond abiraterone: new hormonal therapies for metastatic castration-resistant prostate cancer. Cancer Biol Ther. 2014; 15(2): 149–155. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShahinian VB, Kuo YF, Freeman JL, et al.: Risk of fracture after androgen deprivation for prostate cancer. N Engl J Med. 2005:352(2): 154–164. PubMed Abstract \| Publisher Full Text\n\nSaylor PJ, Lee RJ, Smith MR: Emerging therapies to prevent skeletal morbidity in men with prostate cancer. J Clin Oncol. 2011; 29(27): 3705–3714. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nSaad F, Gleason DM, Murray R, et al.: Long-term efficacy of zoledronic acid for the prevention of skeletal complications in patients with metastatic hormone-refractory prostate cancer. J Natl Cancer Inst. 2004; 96(11): 879–882. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nFizazi K, Carducci M, Smith M, et al.: Denosumab versus zoledronic acid for treatment of bone metastases in men with castration-resistant prostate cancer: a randomised, double-blind study. Lancet. 2011; 377(9768): 813–822. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nSooriakumaran P, Karnes J, Stief C, et al.: A Multi-institutional Analysis of Perioperative Outcomes in 106 Men Who Underwent Radical Prostatectomy for Distant Metastatic Prostate Cancer at Presentation. Eur Urol. 2015; pii: S0302-2838(15)00425-X. PubMed Abstract \| Publisher Full Text\n\nRibas A, Tumeh PC: The future of cancer therapy: selecting patients likely to respond to PD1/L1 blockade. Clin Cancer Res. 2014; 20(19): 4982–4984. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcDermott DF, Atkins MB: PD-1 as a potential target in cancer therapy. Cancer Med. 2013; 2(5): 662–673. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "12477", "date": "16 Feb 2016", "name": "George Huang", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12478", "date": "16 Feb 2016", "name": "Richard J Ablin", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-179
https://f1000research.com/articles/5-178/v1	16 Feb 16	{ "type": "Review", "title": "The role of COP1 in repression of photoperiodic flowering", "authors": [ "Dongqing Xu", "Danmeng Zhu", "Xing Wang Deng", "Dongqing Xu", "Danmeng Zhu" ], "abstract": "Plants use the circadian clock as a timekeeping mechanism to regulate photoperiodic flowering in response to the seasonal changes. CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1), initially identified as a central repressor of seedling photomorphogenesis, was recently shown to be involved in the regulation of light input to the circadian clock, modulating the circadian rhythm and flowering. COP1 encodes a RING-finger E3 ubiquitin ligase and works in concert with SUPPRESSOR of phyA-105 (SPA) proteins to repress photoperiodic flowering by regulating proteasome-mediated degradation of CONSTANS (CO), a central regulator of photoperiodic flowering. In addition, COP1 and EARLY FLOWERING 3 (ELF3) indirectly modulate CO expression via the degradation of GIGANTEA (GI). Here, we summarize the current understanding of the molecular mechanisms underlying COP1’s role in controlling of photoperiodic flowering.", "keywords": [ "COP1", "photoperiodic flowering", "CONSTITUTIVE PHOTOMORPHOGENIC 1", "flowering time" ], "content": "Introduction\n\nIn plants, the phase transition from vegetative to reproductive development is controlled by multiple environmental cues, including photoperiod, light quality, and temperature1. According to their flowering response to the photoperiod change, plants could be classified as long-day (LD) plants, short-day (SD) plants, and day-neutral plants, respectively2. At present, most advances regarding the flowering-time control were obtained in the model facultative LD plant Arabidopsis and the model SD plant rice. A central regulator of LD-induced flowering is the B-box zinc finger transcription factor CONSTANS (CO), which positively regulates flowering time by upregulating the expression of “florigen” FLOWERING LOCUS T (FT) in Arabidopsis3. The control of CO abundance by circadian clock and light plays a crucial role in regulating flowering.\n\nCONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) was initially identified as a key repressor of photomorphogenesis over 20 years ago in Arabidopsis4,5. The subsequent characterization of COP1 revealed its function in multiple light-mediated developmental processes in Arabidopsis and other higher plants, including circadian rhythm and flowering6,7. The ortholog of Arabidopsis COP1 was also found to play vital roles in regulating a variety of developmental processes in animals. COP1 encodes a RING-finger E3 ubiquitin ligase. In Arabidopsis, COP1 functions together with SUPPRESSOR of phyA-105 (SPA) proteins to target the photomorphogenesis-promoting factors for degradation via the 26S proteasome system, such as ELONGATED HYPOCOTYL 5 (HY5), LONG AFTER FAR-RED LIGHT 1 (LAF1), and LONG HYPOCOTYL IN FAR-RED 1 (HFR1)8–11.\n\n\nThe relationship of photoreceptors and COP1 in flowering\n\nIn Arabidopsis, far-red and red light is perceived by phytochromes (phyA-phyE)12,13; blue light is sensed by cryptochromes (CRY1 and CRY2) and several new photoperiodic and/or circadian photoreceptors: ZEITLUPE (ZTL), FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1), and LOV, KELCH PROTEIN 2 (LKP2)14. It was reported that phyA and CRYs are two classes of principal photoperiodic photoreceptors that promote flowering. Mutations in these genes reduce the accumulation of CO protein and delay flowering15,16. During photomorphogenesis, CRYs suppress the activity of the multifunctional E3 ubiquitin ligase COP1 by dissociating the formation of COP1-SPA complex(es), thereby repressing its E3 ubiquitin ligase activity to regulate gene expression in response to blue light17–19. In flowering transition, blue light-dependent CRY2-SPA1 interaction stimulates CRY2-COP1 association to suppress the COP1-dependent proteolysis of CO19. However, how phyA mediates light regulation of protein degradation to modulate developmental timing in flowering is unclear at present. In contrast to cry2, the early-flowering phenotype of phyB in SD is possibly resulting from a COP1-independent mechanism15,16,20. Paradoxically, plants overexpressing phyB also show early flowering, in which the Pfr form of phyB inhibits COP1-SPA activity to stabilize CO and subsequently induce FT expression by phyB-SPA1 direct interaction21.\n\n\nCOP1 direct targets in modulation of flowering\n\nCO acts as a central regulator of photoperiodic flowering, and its abundance directly correlates with the timing of flowering. CO is precisely regulated at both transcriptional and post-translational levels, and this is crucial for Arabidopsis to discriminate the photoperiod and response to light.\n\nThe expression of CO is regulated by circadian clock-associated components, including GIGANTEA (GI), the F-box protein FKF1, and CYCLING DOF FACTORS (CDFs), which regulate daily CO expression profiles22–24. EARLY FLOWERING 3 (ELF3) acts as a substrate adaptor to allow COP1-GI interaction, which leads to the degradation of GI by COP125. FKF1 forms a complex with GI in a light-dependent manner, which contributes to control the CO transcript level by mediating the degradation of CO transcriptional repressors, CDFs22–24. Thus, degradation of GI by COP1 may result in the disassociation of FKF1-GI complex and then negatively regulate CO expression.\n\nPost-translational regulation of CO is another aspect for controlling flowering in response to day length. cop1 mutants display early-flowering phenotype under SD, which is largely related to the change of CO abundance. During the day, CO protein is stabilized, whereas at night CO protein is rapidly degraded through the 26S proteasome pathway mediated by COP1. COP1 directly interacts with the C-terminal of CO in phloem companion cells, where FT protein moves to induce flowering at the shoot apex26,27. In addition, the early-flowering phenotype of spa1 is enhanced by the lesion in SPA3 and SPA4. SPA proteins negatively modulate CO abundance so that spa1 spa3 spa4 triple mutants exhibit strongly increased CO protein levels28. A recent report further demonstrated that the COP1-SPA complex(es) directly interact with the phosphorylated form of CO protein to trigger its protein turnover29.\n\nIn the early morning, TARGET OF EAT (TOE) proteins associate with the transcriptional activation domain of CO to inhibit its activity30. FKE1 stabilizes the CO abundance through a direct interaction in the late afternoon of LD31. At night, CO is degraded through the ubiquitin-mediated 26S proteasome system. Consistently, CO protein levels and its direct target FT peak in the afternoon under LD conditions32. CO activates FT expression mainly through two modes of action: (1) CO directly binds to the CO-responsive element (CORE) in the promoter of FT to activate its expression33. (2) CO physically interacts with two other FT activators NUCLEAR FACTOR-Y (NF-Y) and Myb transcription factor ASYMMETRIC LEAVES 1 (AS1), which directly bind to FT promoter, thus promoting their activation on FT34,35. COP1 triggers the protein turnover of CO, in turn disrupting the formation of CO-NF-Y and CO-AS1 complexes and eventually repressing the FT expression.\n\nBesides light, temperature is another important environmental indicator to determine the appropriate time to flower. Recent work showed that COP1 could act as an integrator of light and cold temperature. cop1 mutants exhibit reduced sensitivity to changes in ambient temperatures in an FT-dependent manner in Arabidopsis. At low ambient temperatures, COP1 is stabilized and subsequently promotes the degradation of GI, which directly activates FT expression to promote flowering36.\n\n\nCOP1-related factors in control of flowering\n\nSimilar to COP1, another repressor of photomorphogenesis, DE-ETIOLATE 1 (DET1), functions as a negative regulator of flowering, as det1 mutants flower early in both LD and SD (extremely early in SD)37. DET1 was shown to be part of the COP10, DE-ETIOLATE 1, DAMAGED DNA-BINDING PROTEIN 1 (CDD) complex, working as CUL4-based E3 ligase38. Co-suppression mutants of CUL4 also showed early-flowering phenotype under SD conditions. CUL4-DDB1 also associates with COP1-SPA complexes39. Together, these studies indicate that a series of E3 ligase complexes may work in concert to repress flowering.\n\nRecent studies revealed that, besides COP1, another RING-finger containing E3 ubiquitin ligase, HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 1 (HOS1), is also involved in controlling the CO protein levels. In the morning of LD, phyB-mediated red light signaling activates HOS1 to degrade CO40. However, on the night of SD, CO protein is ubiquitinated and degraded by COP1-SPA complexes. Consistently, hos1 cop1 double mutants display complete photoperiodic insensitivity, suggesting that HOS1 and COP1 function synergistically in the control of flowering time41,42. Moreover, a regulator of the TOPOISOMERASE VI complex, MIDGET (MID), physically interacts with COP1 and is required for COP1 function as a repressor of flowering under SD conditions43.\n\nIn SD plant rice, PETER PAN SYNDROME (PPS) encodes an ortholog of Arabidopsis COP1. Although PPS is similar to COP1 in repressing photomorphogenesis44, it controls photoperiodic flowering by HEADING DATE 1 (Hd1) (ortholog of Arabidopsis CO) via a currently unknown mechanism45.\n\n\nFuture perspectives\n\nExtensive studies have revealed a complicated but delicate network in regulating photoperiodic flowering in plants. After the role of COP1 in repressing light responses at seedling stage by the regulation of proteolysis was established, later advances have greatly expanded its implication in the control of photoperiodic flowering and circadian rhythm. The studies mentioned in this review have also raised a number of challenging questions to be addressed in the future. As a long-term goal, the roles of COP1 in light quality control of flowering would be of great interest to determine. Specifically, how does COP1 work in concert or function antagonistically with other key factors to control CO abundance/activity in a special photoperiod or in response to multiple environmental cues? How does COP1 determine the substrates to be degraded by the COP1–SPA complex alone or together by other COP/DET/FUS protein-containing complex(es)? Moreover, the identification and characterization of novel direct targets of COP1 in the control of photoperiodic flowering will assist us in understanding the molecular mechanism underlying CO-independent pathways. In addition, further studies on the differential mechanisms of COP1 function in Arabidopsis and crop plants will help us to explore their functional novelty and diversity during the evolution of monocots and dicots.\n\n\nAbbreviations\n\nAS1, ASYMMETRIC LEAVES 1; CDF, CYCLING DOF FACTOR; CO, CONSTANS; COP1, CONSTITUTIVE PHOTOMORPHOGENIC 1; CRY, cryptochromes; CUL4, CULLIN4; DET1, DE-ETIOLATE 1; FKF1, FLAVIN BINDING, KELCH REPEAT, F-BOX 1; FT, FLOWERING LOCUS T; GI, GIGANTEA; HOS1, HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 1; LD, long-day; MID, MIDGET; PPS, PETER PAN SYNDROME; NF-Y, NUCLEAR FACTOR-Y; PHY, phytochromes; SD, short-day; SPA, SUPPRESSOR of phyA-105.", "appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThis work was supported by the National Natural Science Foundation of China (31330048) and the National Institutes of Health (GM047850).\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nWe apologize to the colleagues whose work could not be cited because of space limitations.\n\n\nReferences\n\nRomera-Branchat M, Andrés F, Coupland G: Flowering responses to seasonal cues: what's new? 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PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nZhu D, Maier A, Lee JH, et al.: Biochemical characterization of Arabidopsis complexes containing CONSTITUTIVELY PHOTOMORPHOGENIC1 and SUPPRESSOR OF PHYA proteins in light control of plant development. Plant Cell. 2008; 20(9): 2307–23. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBae G, Choi G: Decoding of light signals by plant phytochromes and their interacting proteins. Annu Rev Plant Biol. 2008; 59: 281–311. PubMed Abstract \| Publisher Full Text\n\nChen M, Chory J: Phytochrome signaling mechanisms and the control of plant development. Trends Cell Biol. 2011; 21(11): 664–71. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu H, Liu B, Zhao C, et al.: The action mechanisms of plant cryptochromes. Trends Plant Sci. 2011; 16(12): 684–91. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nValverde F, Mouradov A, Soppe W, et al.: Photoreceptor regulation of CONSTANS protein in photoperiodic flowering. Science. 2004; 303(5660): 1003–6. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nMockler TC, Guo H, Yang H, et al.: Antagonistic actions of Arabidopsis cryptochromes and phytochrome B in the regulation of floral induction. Development. 1999; 126(10): 2073–82. PubMed Abstract \| F1000 Recommendation\n\nLian HL, He SB, Zhang YC, et al.: Blue-light-dependent interaction of cryptochrome 1 with SPA1 defines a dynamic signaling mechanism. Genes Dev. 2011; 25(10): 1023–8. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nLiu B, Zuo Z, Liu H, et al.: Arabidopsis cryptochrome 1 interacts with SPA1 to suppress COP1 activity in response to blue light. Genes Dev. 2011; 25(10): 1029–34. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nZuo Z, Liu H, Liu B, et al.: Blue light-dependent interaction of CRY2 with SPA1 regulates COP1 activity and floral initiation in Arabidopsis. Curr Biol. 2011; 21(10): 841–7. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nReed JW, Nagatani A, Elich TD, et al.: Phytochrome A and Phytochrome B Have Overlapping but Distinct Functions in Arabidopsis Development. Plant Physiol. 1994; 104(4): 1139–49. PubMed Abstract \| Free Full Text\n\nHajdu A, Ádám É, Sheerin DJ, et al.: High-level expression and phosphorylation of phytochrome B modulates flowering time in Arabidopsis. Plant J. 2015; 83(5): 794–805. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nImaizumi T, Schultz TF, Harmon FG, et al.: FKF1 F-box protein mediates cyclic degradation of a repressor of CONSTANS in Arabidopsis. Science. 2005; 309(5732): 293–7. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nSawa M, Nusinow DA, Kay SA, et al.: FKF1 and GIGANTEA complex formation is required for day-length measurement in Arabidopsis. Science. 2007; 318(5848): 261–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFornara F, Panigrahi KC, Gissot L, et al.: Arabidopsis DOF transcription factors act redundantly to reduce CONSTANS expression and are essential for a photoperiodic flowering response. Dev Cell. 2009; 17(1): 75–86. PubMed Abstract \| Publisher Full Text\n\nYu JW, Rubio V, Lee NY, et al.: COP1 and ELF3 control circadian function and photoperiodic flowering by regulating GI stability. Mol Cell. 2008; 32(5): 617–30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJang S, Marchal V, Panigrahi KC, et al.: Arabidopsis COP1 shapes the temporal pattern of CO accumulation conferring a photoperiodic flowering response. EMBO J. 2008; 27(8): 1277–88. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu LJ, Zhang YC, Li QH, et al.: COP1-mediated ubiquitination of CONSTANS is implicated in cryptochrome regulation of flowering in Arabidopsis. Plant Cell. 2008; 20(2): 292–306. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nLaubinger S, Marchal V, Le Gourrierec J, et al.: Arabidopsis SPA proteins regulate photoperiodic flowering and interact with the floral inducer CONSTANS to regulate its stability. Development. 2006; 133(16): 3213–22. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nSarid-Krebs L, Panigrahi KC, Fornara F, et al.: Phosphorylation of CONSTANS and its COP1-dependent degradation during photoperiodic flowering of Arabidopsis. Plant J. 2015; 84(3): 451–63. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nZhang B, Wang L, Zeng L, et al.: Arabidopsis TOE proteins convey a photoperiodic signal to antagonize CONSTANS and regulate flowering time. Genes Dev. 2015; 29(9): 975–87. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nSong YH, Smith RW, To BJ, et al.: FKF1 conveys timing information for CONSTANS stabilization in photoperiodic flowering. Science. 2012; 336(6084): 1045–9. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nSalazar JD, Saithong T, Brown PE, et al.: Prediction of photoperiodic regulators from quantitative gene circuit models. Cell. 2009; 139(6): 1170–9. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nTiwari SB, Shen Y, Chang HC, et al.: The flowering time regulator CONSTANS is recruited to the FLOWERING LOCUS T promoter via a unique cis-element. New Phytol. 2010; 187(1): 57–66. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nWenkel S, Turck F, Singer K, et al.: CONSTANS and the CCAAT box binding complex share a functionally important domain and interact to regulate flowering of Arabidopsis. Plant Cell. 2006; 18(11): 2971–84. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nSong YH, Lee I, Lee SY, et al.: CONSTANS and ASYMMETRIC LEAVES 1 complex is involved in the induction of FLOWERING LOCUS T in photoperiodic flowering in Arabidopsis. Plant J. 2012; 69(2): 332–42. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nJang K, Lee HG, Jung SJ, et al.: The E3 Ubiquitin Ligase COP1 Regulates Thermosensory Flowering by Triggering GI Degradation in Arabidopsis. Sci Rep. 2015; 5: 12071. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nKang MY, Yoo SC, Kwon HY, et al.: Negative regulatory roles of DE-ETIOLATED1 in flowering time in Arabidopsis. Sci Rep. 2015; 5: 9728. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nChen H, Shen Y, Tang X, et al.: Arabidopsis CULLIN4 Forms an E3 Ubiquitin Ligase with RBX1 and the CDD Complex in Mediating Light Control of Development. Plant Cell. 2006; 18(8): 1991–2004. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen H, Huang X, Gusmaroli G, et al.: Arabidopsis CULLIN4-damaged DNA binding protein 1 interacts with CONSTITUTIVELY PHOTOMORPHOGENIC1-SUPPRESSOR OF PHYA complexes to regulate photomorphogenesis and flowering time. Plant Cell. 2010; 22(1): 108–23. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLazaro A, Valverde F, Piñeiro M, et al.: The Arabidopsis E3 ubiquitin ligase HOS1 negatively regulates CONSTANS abundance in the photoperiodic control of flowering. Plant Cell. 2012; 24(3): 982–99. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nJung JH, Seo PJ, Park CM: The E3 ubiquitin ligase HOS1 regulates Arabidopsis flowering by mediating CONSTANS degradation under cold stress. J Biol Chem. 2012; 287(52): 43277–87. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nJung JH, Lee HJ, Park MJ, et al.: Beyond ubiquitination: proteolytic and nonproteolytic roles of HOS1. Trends Plant Sci. 2014; 19(8): 538–45. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nSchrader A, Uhrig J: MIDGET cooperates with COP1 and SPA1 to repress flowering in Arabidopsis thaliana. Plant Signal Behav. 2013; 8(9): pii: e25600. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nTsuge T, Inagaki N, Yoshizumi T, et al.: Phytochrome-mediated control of COP1 gene expression in rice plants. Mol Genet Genomics. 2001; 265(1): 43–50. PubMed Abstract \| Publisher Full Text\n\nTanaka N, Itoh H, Sentoku N, et al.: The COP1 ortholog PPS regulates the juvenile-adult and vegetative-reproductive phase changes in rice. Plant Cell. 2011; 23(6): 2143–54. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation" }	[ { "id": "12469", "date": "16 Feb 2016", "name": "Haiyang Wang", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12470", "date": "16 Feb 2016", "name": "Nam-Chon Paek", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-178
https://f1000research.com/articles/5-177/v1	16 Feb 16	{ "type": "Review", "title": "Advances in Understanding and Managing Chronic Urticaria", "authors": [ "Yasmin Moolani", "Charles Lynde", "Gordon Sussman", "Yasmin Moolani", "Charles Lynde" ], "abstract": "There have been recent advances in the classification and management of chronic urticaria. The new term chronic spontaneous urticaria (CSU) has replaced chronic idiopathic urticaria and chronic autoimmune urticaria. In addition, chronic inducible urticaria (CINDU) has replaced physical urticaria and includes other forms of inducible urticaria, such as cholinergic and aquagenic urticaria. Furthermore, novel research has resulted in a new understanding with guidelines being revised in the past year by both the American Academy of Allergy, Asthma, and Immunology (AAAAI) and the European Academy of Allergy and Clinical Immunology (EAACI)/Global Allergy and Asthma European Network (GA2LEN)/European Dermatology Forum (EDF)/World Allergy Organization (WAO). There are some differences in the recommendations, which will be discussed, but the core updates are common to both groups. The basic treatment for chronic urticaria involves second-generation non-sedating non-impairing H1 antihistamines as first-line treatment. This is followed by up to a 4-fold increase in the licensed dose of these H1 antihistamines. The major therapeutic advance in recent years has been in third-line treatment with omalizumab, a humanized monoclonal anti-immunoglobulin E (anti-IgE) antibody that prevents binding of IgE to the high-affinity IgE receptor. Several multicenter randomized controlled trials have shown safety and efficacy of omalizumab for CSU. There are also some small studies showing efficacy of omalizumab in CINDU. While there were previously many treatment options which were lacking in strong evidence, we are moving into an era where the treatment algorithm for chronic urticaria is simplified and contains more evidence-based, effective, and less toxic treatment options.", "keywords": [ "Chronic Urticaria", "Classification", "Management", "Antihistamines", "Up-dosing", "Omalizumab" ], "content": "Introduction\n\nUrticaria (hives) is a relatively common condition, with a point prevalence of about 0.5–1%1. The peak incidence is in the range of 20–40 years. Urticaria is the general term for a cutaneous response characterized by wheals and swellings. A deeper localized swelling often associated with urticaria is called angioedema. Urticaria is mediated by mast cell degranulation. Mast cells can be activated by immunologic and non-immunologic mechanisms, which lead to degranulation of inflammatory mediators including histamine, leukotrienes, and prostaglandins. Release of these mediators causes the characteristic pruritus, vascular permeability, and edema.\n\n\nClassification\n\nAcute urticaria is defined as hives that last less than 6 weeks, while chronic urticaria refers to urticaria that occurs intermittently for at least 6 weeks, typically on most days of the week. Acute urticaria can occur spontaneously or in response to a trigger. Triggers of acute urticaria are typically acute viral infections or allergic reactions to foods, medications, latex, or insects. Chronic urticaria can also be spontaneous or inducible, though the triggers of inducible urticaria are different compared to those of acute urticaria. Inducible urticaria involves hiving responses resulting from physical stimuli including scratch (dermographism) and cold contact urticaria. Additional triggers of chronic urticaria include pressure, temperature change, sun exposure, water exposure, and exercise (Figure 1, Table 1)2–5.\n\nThe revised classification of urticaria divides chronic urticaria into chronic spontaneous urticaria (CSU), with no external trigger, and chronic inducible urticaria (CINDU), with an identifiable trigger. Acute urticaria remains as that which lasts less than 6 weeks.\n\nThe triggers of acute and chronic urticaria are different and are outlined in this table.\n\n\nNew terminology\n\nWhile the pathogenesis of urticaria remains incompletely understood, our knowledge of the etiology of chronic urticaria has increased. We are able to differentiate the older terminology of chronic autoimmune urticaria or chronic idiopathic urticaria from physically induced urticaria6. However, now physical urticaria has been revised to chronic inducible urticaria (CINDU) to reflect the external trigger and inducible nature. The term CINDU also includes cholinergic, aquagenic, and contact urticaria. The remaining forms of urticaria, which occur without an external trigger and instead via an endogenous mechanism, are classified as chronic spontaneous urticaria (CSU). This is simplified and more specific than the term chronic idiopathic urticaria, which is now falling out of use2 (Figure 1).\n\n\nAssessing disease severity and impact on quality of life\n\nThe primary symptom of urticaria is pruritus. Urticaria generally causes significant disability adversely affecting an individual’s life. New tools have been developed to quantify the effect of urticaria on quality of life. One such tool is the Urticaria Activity Score (UAS7), which is a validated score recommended by urticaria guidelines7. In the UAS7, the patient records the severity of itching and the number of wheals daily for 7 days. A score of <7 in 1 week indicates control of disease, whereas a score of >28 per week indicates severe disease. This tool allows for efficient clinical practice, maximizing the information gathered during patient visits while minimizing the use of resources and time. While the UAS7 is a prospective tool, a retrospective tool was also developed, the Urticaria Control Test (UCT)8. Additional questionnaires have been developed and are recommended for use to assess the impact of urticaria on quality of life. The recently developed tools include the Chronic Urticaria Quality of Life Questionnaire (CU-Q2oL)9 and the Urticaria Severity Score10. These tools were designed specifically for urticaria since other more generic questionnaires do not capture the nuances of the impact of urticaria on one’s quality of life (Table 2).\n\nThere are a number of recently developed tools specifically for urticaria to assess severity and impact on quality of life.\n\n\nAdvances in management\n\nRecently, new guidelines have been published by the American Academy of Allergy, Asthma, and Immunology (AAAAI) and the European Academy of Allergy and Clinical Immunology (EAACI)/Global Allergy and Asthma European Network (GA2LEN)/European Dermatology Forum (EDF)/World Allergy Organization (WAO) that update what is currently understood about urticaria3–5. There are several other published regional guidelines with similar recommendations, including Canadian11. With the exception of the American guidelines, the other organizations put forth a similar algorithm. That is, the following stepwise approach: first-line treatment includes second-generation H1 antihistamines, second-line therapy involves up-dosing the second-generation H1 antihistamine, and third-line treatment includes a new medication, omalizumab, which we recommend to be used before a more toxic medication, cyclosporine A.\n\nOne of the notable differences in the guidelines put forth by the American versus European/World guidelines is the role for first-generation H1 antihistamines. The AAAAI keeps first-generation H1 antihistamines in the treatment algorithm. The EAACI/GA2LEN/EDF/WAO guidelines specifically recommend avoiding first-generation H1 antihistamines based on the benefit-to-risk ratio of these agents. The known risks are cited as well as the high underestimated risk potential when these agents are taken in the evening, such as changes in REM sleep patterns and impairment of cognitive functions12. For similar reasons, the EAACI/GA2LEN/EDF/WAO guidelines do not address a role for hydroxyzine or doxepin, whereas the AAAAI guidelines include these medications as options if up-dosing of non-sedating, non-impairing H1 antihistamines is not successful at complete control of symptoms.\n\nThe EAACI/GA2LEN/EDF/WAO guidelines do not include H2 antihistamines in their algorithm due to a recent Cochrane review that shows lack of evidence of efficacy of these medications13. Thus, H2 antihistamines are advised only on an individual case basis but not as first-, second- or third-line treatment. The AAAAI guidelines, however, consider the options of up-dosing second-generation H1 antihistamines, adding other second-generation H1 antihistamines, and adding H2 antagonists, leukotriene receptor antagonists or first-generation H1 antihistamines at bedtime to all be equally weighted second-line options3. In doing this, however, the adverse reaction profile would increase.\n\nBoth guidelines recommend that corticosteroids should be considered only for the short-term intervention and avoided as long-term treatments due to the significant number of side effects and alternative options for treatment. Both guidelines also acknowledge a role for cyclosporine A as an add-on treatment for patients who have refractory chronic urticaria that is not responsive to the above mentioned treatments3,5.\n\nBoth guidelines have also incorporated a new third-line treatment option, omalizumab, a subcutaneously injected, biologic medication3,5. There is a large body of new data showing efficacy of omalizumab in various subtypes of chronic urticaria with a relatively low level of adverse effects18–24. Omalizumab is a recombinant humanized immunioglobulin G1 (IgG1) monoclonal antibody that binds to IgE. In binding IgE, omalizumab inhibits binding of IgE to the high-affinity IgE receptor (FcεRI) found on the surface of mast cells and basophils. By inhibiting binding of IgE to the receptor, the release of inflammatory mediators such as histamine, leukotrienes, and prostaglandins is limited, thus blunting the inflammatory response14. By inhibiting IgE from binding the FcεRI receptor, omalizumab also has the effect of down-regulating the FcεRI receptor on the surface of mast cells and basophils. This monoclonal antibody has also been shown to decrease the release of circulating interleukin-6 and tumor necrosis factor α and to decrease the recruitment of T cells, eosinophils, and macrophages in the inflammatory response15. The overall effect of omalizumab is to prevent urticaria and angioedema14.\n\nOmalizumab was approved in Europe and North America in 2014 for the treatment of CSU in adults and adolescents 12 years of age and older who do not respond to H1 antihistamines16,17. This approval was based on three phase III randomized clinical trials evaluating over 1000 patients’ treatment response with inadequately controlled CSU18–20. There have also been several published real life trials demonstrating efficacy and safety21–23. Additionally, there are smaller case studies assessing the efficacy of omalizumab in various subtypes of CINDU, and while the number of overall cases is low, all have shown notable improvement in urticaria symptoms24. Future research is, therefore, required to evaluate the role of omalizumab in the various subtypes of chronic urticaria as well as to establish standardized protocols for dosing and monitoring adverse effects of long-term therapy.\n\n\nConclusion\n\nA number of notable changes have occurred recently in the guidelines for managing chronic urticaria. Overall, the classification and management have become more simplified. There are new tools to track and monitor the quality of life of patients with this challenging disease. Furthermore, the recent addition of omalizumab to the treatment algorithm for CSU has offered a more effective and less toxic treatment option to those suffering with refractory disease to older treatment strategies. With the many trials currently underway for CINDU, the outlook is optimistic.", "appendix": "Competing interests\n\n\n\nGordon Sussman is a consultant, speaker, and researcher for Novartis pharmaceuticals. He is also an advisor for CSL Behring and Merck and does research for DBV Technologies, Merck, and AstraZeneca.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nMaurer M, Weller K, Bindslev-Jensen C, et al.: Unmet clinical needs in chronic spontaneous urticaria. A GA²LEN task force report. Allergy. 2011; 66(3): 317–330. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nMaurer M, Bindslev-Jensen C, Gimenez-Arnau A, et al.: Chronic idiopathic urticaria (CIU) is no longer idiopathic: time for an update. Br J Dermatol. 2013; 168(2): 455–456. PubMed Abstract \| Publisher Full Text\n\nBernstein JA, Lang DM, Khan DA, et al.: The diagnosis and management of acute and chronic urticaria: 2014 update. J Allergy Clin Immunol. 2014; 133(5): 1270–1277. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nZuberbier T, Aberer W, Asero R, et al.: Methods report on the development of the 2013 revision and update of the EAACI/GA2LEN/EDF/WAO guideline for the definition, classification, diagnosis, and management of urticaria. Allergy. 2014; 69(7): e1–29. PubMed Abstract \| Publisher Full Text\n\nZuberbier T, Aberer W, Asero R, et al.: The EAACI/GA2LEN/EDF/WAO Guideline for the definition, classification, diagnosis, and management of urticaria: the 2013 revision and update. Allergy. 2014; 69(7): 868–887. PubMed Abstract \| Publisher Full Text\n\nKaplan AP: Chronic urticaria: pathogenesis and treatment. J Allergy Clin Immunol. 2004; 114(3): 465–74, quiz 475. PubMed Abstract \| Publisher Full Text\n\nMłynek A, Zalewska-Janowska A, Martus P, et al.: How to assess disease activity in patients with chronic urticaria? Allergy. 2008; 63(6): 777–780. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nWeller K, Groffik A, Church MK, et al.: Development and validation of the Urticaria Control Test: a patient-reported outcome instrument for assessing urticaria control. J Allergy Clin Immunol. 2014; 133(5): 1365–72, 1372.e1–6. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nBaiardini I, Pasquali M, Braido F, et al.: A new tool to evaluate the impact of chronic urticaria on quality of life: chronic urticaria quality of life questionnaire (CU-QoL). Allergy. 2005; 60(8): 1073–1078. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nJariwala SP, Moday H, de Asis ML, et al.: The Urticaria Severity Score: a sensitive questionnaire/index for monitoring response to therapy in patients with chronic urticaria. Ann Allergy Asthma Immunol. 2009; 102(6): 475–482. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nSussman G, Hébert J, Gulliver W, et al.: Insights and advances in chronic urticaria: a Canadian perspective. Allergy Asthma Clin Immunol. 2015; 11(1): 7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStaevska M, Gugutkova M, Lazarova C, et al.: Night-time sedating H1 -antihistamine increases daytime somnolence but not treatment efficacy in chronic spontaneous urticaria: a randomized controlled trial. Br J Dermatol. 2014; 171(1): 148–154. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nFedorowicz Z, van Zuuren EJ, Hu N: Histamine H2-receptor antagonists for urticaria. Cochrane Database Syst Rev. 2012; 3: CD008596. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nXolair® Omalizumab. Fda.gov. United States Food and Drug Administration. 2015. Reference Source\n\nChang TW, Chen C, Lin CJ, et al.: The potential pharmacologic mechanisms of omalizumab in patients with chronic spontaneous urticaria. J Allergy Clin Immunol. 2015; 135(2): 337–342. PubMed Abstract \| Publisher Full Text\n\nNovartis Institutes for BioMedical Research: \"Novartis Announces US FDA Approval of Xolair® for Chronic Idiopathic Urticaria (CIU).\" Novartis Media Relations, 2014. Reference Source\n\nNovartis.ca. Novartis Pharmaceuticals Canada Inc.: \"Novartis Announces That Xolair® Is Approved in Canada as a Licensed Therapy for Chronic Idiopathic Urticaria (CIU) Patients Unresponsive to Antihistamines.\" 2014. Reference Source\n\nMaurer M, Rosén K, Hsieh HJ, et al.: Omalizumab for the treatment of chronic idiopathic or spontaneous urticaria. N Engl J Med. 2013; 368(10): 924–935. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nKaplan A, Ledford D, Ashby M, et al.: Omalizumab in patients with symptomatic chronic idiopathic/spontaneous urticaria despite standard combination therapy. J Allergy Clin Immunol. 2013; 132(1): 101–109. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nSaini SS, Bindslev-Jensen C, Maurer M, et al.: Efficacy and safety of omalizumab in patients with chronic idiopathic/spontaneous urticaria who remain symptomatic on H1 antihistamines: a randomized, placebo-controlled study. J Invest Dermatol. 2015; 135(1): 67–75. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nSussman G, Hébert J, Barron C, et al.: Real-life experiences with omalizumab for the treatment of chronic urticaria. Ann Allergy Asthma Immunol. 2014; 112(2): 170–174. PubMed Abstract \| Publisher Full Text\n\nMetz M, Ohanyan T, Church MK, et al.: Omalizumab is an effective and rapidly acting therapy in difficult-to-treat chronic urticaria: a retrospective clinical analysis. J Dermatol Sci. 2014; 73(1): 57–62. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nLabrador-Horrillo M, Valero A, Velasco M, et al.: Efficacy of omalizumab in chronic spontaneous urticaria refractory to conventional therapy: analysis of 110 patients in real-life practice. Expert Opin Biol Ther. 2013; 13(9): 1225–1228. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nMaurer M, Church MK, Gonçalo M, et al.: Management and treatment of chronic urticaria (CU). J Eur Acad Dermatol Venereol. 2015; 29(Suppl 3): 16–32. PubMed Abstract \| Publisher Full Text" }	[ { "id": "12467", "date": "16 Feb 2016", "name": "Richard Warrington", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12468", "date": "16 Feb 2016", "name": "Riccardo Asero", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-177
https://f1000research.com/articles/5-175/v1	16 Feb 16	{ "type": "Opinion Article", "title": "A new hypothesis: some metastases are the result of inflammatory processes by adapted cells, especially adapted immune cells at sites of inflammation", "authors": [ "Leili Shahriyari" ], "abstract": "There is an old hypothesis that metastasis is the result of migration of tumor cells from the tumor to a distant site. In this article, we propose another mechanism for metastasis, for cancers that are initiated at the site of chronic inflammation. We suggest that cells at the site of chronic inflammation might become adapted to the inflammatory process, and these adaptations may lead to the initiation of an inflammatory tumor. For example, in an inflammatory tumor immune cells might be adapted to send signals of proliferation or angiogenesis, and epithelial cells might be adapted to proliferation (like inactivation of tumor suppressor genes). Therefore, we hypothesize that metastasis could be the result of an inflammatory process by adapted cells, especially adapted immune cells at the site of inflammation, as well as the migration of tumor cells with the help of activated platelets, which travel between sites of inflammation. If this hypothesis is correct, then any treatment causing necrotic cell death may not be a good solution. Because necrotic cells in the tumor micro-environment or anywhere in the body activate the immune system to initiate the inflammatory process, and the involvement of adapted immune cells in the inflammatory processes leads to the formation and progression of tumors. Adapted activated immune cells send more signals of proliferation and/or angiogenesis than normal cells. Moreover, if there were adapted epithelial cells, they would divide at a much higher rate in response to the proliferation signals than normal cells. Thus, not only would the tumor come back after the treatment, but it would also grow more aggressively.", "keywords": [ "Metastasis", "Cancer", "Chronic inflammation", "Adapted immune cells", "Inflammatory processes", "Immune system", "Platelets", "Wound healing process." ], "content": "\n\nMany cancers arise from sites of chronic inflammation1. Immune cells inside the chronic inflammation site initiate tumor progression by releasing reactive oxygen or nitrogen species, which lead to DNA damage in epithelial cells2. Inflammation not only can cause mutation in epithelial cells3, but can also change their fitness4.\n\nIn chronic inflammation, T-cells might become adapted to send high levels of proliferation signals, and regulatory T-cells might have been changed to prevent their inhibition5. Effector T-cells also create an environment for tumor initiation and progression by releasing tumor-promoting cytokines IL-62.\n\nThese findings suggest that cells at the site of chronic inflammation are adapted to the wound healing process. Immune cells are adapted to send signals of proliferation or angiogenesis, and tissue cells are adapted to proliferation (like inactivation of tumor suppressor genes). These adaptations lead to the initiation of a tumor.\n\nIf there are adapted immune cells, then we can look at metastasis from a new perspective. Any site of inflammation might recruit adapted immune cells, and then they contribute to the inflammatory process there. If immune cells are adapted to send more signals of proliferation and angiogenesis, then new tumors would initiate at sites of inflammation. Below we gather some evidence that supports this hypothesis.\n\n• There is a hypothesis that metastasis is the result of cancer cell migration from the tumor extracellular matrix to the bloodstream or lymphatic vessels as circulating tumor cells (CTCs), and then to the new site. CTCs only use lymphatic routes to migrate to the nearby lymph nodes, but not for traveling long distances6. Cell migration occurs when tumor epithelial (E) cells lose their cell-cell adhesion and become motile mesenchymal (M) cells, i.e. epithelial-mesenchymal transition (EMT). When these CTCs exit the bloodstream, they undergo a reverse process called mesenchymal-epithelial transition (MET) to continue their differentiation and develop a secondary tumor7–9.\n\nIn inflammatory breast cancer, there is a correlation between immune activation and CTCs with EMT characteristics10. Cohen et al. hypothesized that EMT could be the result of inflammatory processes initiated by activated immune cells10. Epithelial cells from the colonic epithelium of patients with benign colon diseases also circulate in the blood11. During wound healing, epithelial cells migrate and disperse as individual mesenchymal cells by down regulating cell-cell junctions12. Thus, CTCs in blood could be the result of inflammation.\n\n• By querying published available data sets13–17, we calculate the probability of not detecting any CTCs in blood from patients with metastatic breast cancer, and the result is 0.6. That means there might be other phenomena, beside CTCs, that cause metastasis. Since, no CTCs were detected in the blood of 29% of metastatic breast cancer patients starting a first or new line of therapy, it is unlikely that treatments are responsible for not observing CTCs in blood16.\n\n• CTC-clusters are rare compared with single CTCs, however CTC-clusters, which usually include platelets and white blood cells18, significantly increase metastatic potential19. Moreover inhibition of platelet activation or platelet depletion decreases metastasis rates20–22. There is a hypothesis that metastasis is reduced when the host is platelet depleted because platelets are protecting tumor cells from natural killing cells23,24 Platelets in tumor cell clusters release transforming growth factor β (TGF-β)25, and tumor cells that are bound to platelets highly express EMT-associated genes26.\n\n• Cancer of unknown primary origin (CUP) is defined by the presence of metastatic disease with no identified primary tumor. CUPs are 3.5% of all human malignancies, and one of the ten most frequent cancers worldwide27. The major sites of CUP are bones, liver, lung, and lymph nodes28, the sites where immune cells are most active.\n\n• New studies show that recruited bone marrow progenitor cells change the host’s environment to generate the “pre-metastatic niche” to which the tumor cells metastasize29. Bone marrow-derived haematopoietic progenitor cells (HPCs) that express vascular endothelial growth factor receptor 1 form cellular clusters at pre-metastatic sites before appearance of a single tumor cell30.\n\n• There is evidence of metastasis to the site of injury. Two patients with squamous cell carcinoma of the lung developed distant localized metastatic disease at sites of physical injuries; one to the knee injured in an accidental fall six weeks earlier, and the other to portions of the liver injured in a mechanical fall two months earlier31. In a mice model of metastatic breast cancer, radiation-induced pulmonary injury lead to chronic inflammatory responses, and development of pre-metastatic niches32. In another mice model, hepatic ischemia-reperfusion injury increased the number of liver metastases of human pancreatic cancer (Capan-1) cells, which were injected into the mice spleen33. Several studies show that lung injury induced by the chemotherapy drug, bleomycin, increases lung metastases; they also observed tumor cell adherence to extracellular matrix and fibrin at injured areas34,35. Therefore we suggest that the sites of injuries are potential metastatic sites.\n\nWe hypothesize that chronic inflammation can cause adapted bone marrow derived cells (for example, adapted macrophages or T-cells) and/or adapted tissue cells (for instance, adapted epithelial cells or stromal fibroblasts) to lead tumor initiation and progression. If adapted immune cells are present, then the new site of inflammation might recruit these adapted immune cells and cause metastasis. Additionally, the new site of inflammation may recruit activated platelets. The activated platelets travel between sites of inflammation, including the site of inflammatory carcinoma. Tumor cells can link to adhesion receptors on platelets and travel to the new site of inflammation. The activated platelets start the wound healing process at the new site, which now includes some tumor cells. As the tumor cells respond to the wound healing signals more strongly than normal cells, new tumors would initiate in the site of inflammation (Figure 1).\n\nChronic inflammation leads to adapted tissue and/or adapted immune cells. These adaptations cause tumor initiation. A new inflammation site recruits these adapted immune cells. The adapted immune cells start the wound healing process at the inflammation site and send more inflammatory signals than normal immune cells. Thus, a new tumor initiates there. Also, the activated platelets travel between sites of inflammation, including the site of primary inflammatory tumor. Tumor cells can link to adhesion receptors on platelets and travel to the new site of inflammation with the help of platelets. The activated platelets start the inflammatory process at the new site, which now includes some tumor cells. Tumor cells respond to the inflammatory signals more strongly than normal cells. The transported tumor cells initiate a tumor at the inflammation site.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis research has been supported in part by the Mathematical Biosciences Institute and the National Science Foundation under grant DMS 0931642.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nBalkwill F, Charles KA, Mantovani A: Smoldering and polarized inflammation in the initiation and promotion of malignant disease. Cancer Cell. 2005; 7(3): 211–217. PubMed Abstract \| Publisher Full Text\n\nWaldner MJ, Neurath MF: Colitis-associated cancer: the role of T cells in tumor development. Semin Immunopathol. 2009; 31(2): 249–256. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTzanakakis GN, Agarwal KC, Veronikis DK, et al.: Effects of antiplatelet agents alone or in combinations on platelet aggregation and on liver metastases from a human pancreatic adenocarcinoma in the nude mouse. J Surg Oncol. 1991; 48(1): 45–50. PubMed Abstract \| Publisher Full Text\n\nAmirkhosravi A, Mousa SA, Amaya M, et al.: Inhibition of tumor cell-induced platelet aggregation and lung metastasis by the oral GpIIb/IIIa antagonist XV454. Thromb Haemost. 2003; 90(3): 549–554. PubMed Abstract \| Publisher Full Text\n\nPalumbo JS, Talmage KE, Massari JV, et al.: Platelets and fibrin(ogen) increase metastatic potential by impeding natural killer cell-mediated elimination of tumor cells. Blood. 2005; 105(1): 178–185. PubMed Abstract \| Publisher Full Text\n\nPalumbo JS: Mechanisms linking tumor cell-associated procoagulant function to tumor dissemination. Semin Thromb Hemost. 2008; 34(2): 154–160. PubMed Abstract \| Publisher Full Text\n\nNieswandt B, Hafner M, Echtenacher B, et al.: Lysis of tumor cells by natural killer cells in mice is impeded by platelets. Cancer Res. 1999; 59(6): 1295–1300. PubMed Abstract\n\nYu M, Bardia A, Wittner BS, et al.: Circulating breast tumor cells exhibit dynamic changes in epithelial and mesenchymal composition. Science. 2013; 339(6119): 580–584. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLabelle M, Begum S, Hynes RO: Direct signaling between platelets and cancer cells induces an epithelial-mesenchymal-like transition and promotes metastasis. Cancer Cell. 2011; 20(5): 576–590. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPavlidis N, Fizazi K: Carcinoma of unknown primary (CUP). Crit Rev Oncol Hematol. 2009; 69(3): 271–278. PubMed Abstract \| Publisher Full Text\n\nHainsworth JD, Greco FA: Cancer of Unknown Primary Origin. Goldman’s Cecil Medicine: Twenty Fourth Edition. 2012; 1: 1334–1337. Publisher Full Text\n\nPeinado H, Lavotshkin S, Lyden D: The secreted factors responsible for pre-metastatic niche formation: old sayings and new thoughts. Semin Cancer Biol. 2011; 21(2): 139–146. PubMed Abstract \| Publisher Full Text\n\nKaplan RN, Riba RD, Zacharoulis S, et al.: VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche. Nature. 2005; 438(7069): 820–827. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWalter ND, Rice PL, Redente EF, et al.: Wound healing after trauma may predispose to lung cancer metastasis: review of potential mechanisms. Am J Respir Cell Mol Biol. 2010; 44(5): 591–6. PubMed Abstract \| Publisher Full Text\n\nGong HY, Hu WG, Hu QY, et al.: Radiation-induced pulmonary injury accelerated pulmonary metastasis in a mouse model of breast cancer. Oncol Lett. 2015; 10(6): 3613–3618. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYoshimoto K, Tajima H, Ohta T, et al.: Increased E-selectin in hepatic ischemia-reperfusion injury mediates liver metastasis of pancreatic cancer. Oncol Rep. 2012; 28(3): 791–796. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOrr FW, Adamson IY, Young L: Promotion of pulmonary metastasis in mice by bleomycin-induced endothelial injury. Cancer Res. 1986; 46(2): 891–7. PubMed Abstract\n\nAdamson IY, Orr FW, Young L: Effects of injury and repair of the pulmonary endothelium on lung metastasis after bleomycin. J Pathol. 1986; 150(4): 279–87. PubMed Abstract \| Publisher Full Text" }	[ { "id": "13279", "date": "18 Apr 2016", "name": "Maryam Keshtkar Jahromi", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 hypothesis has a potential to make a positive impact in cancer research and drug discovery. For cancer patients, it might help to accelerate both the development of promising therapies and the potential of personalized medicine — treatments based on an individual’s unique immune deficiency. This is a very interesting and unique idea which needs more research.", "responses": [] }, { "id": "13492", "date": "22 Apr 2016", "name": "Patrice Forget", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe inflammatory nature of cancer, and especially metastases development, becomes a paradigm shift. This text summarizes well some new hypotheses and future challenges. Next steps to translate this in clinical trials are then needed.", "responses": [] }, { "id": "13490", "date": "27 Apr 2016", "name": "Alain Borgeat", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 more and more evidence that cancer metastases and inflammation share a common pathway. Therefore this hypothesis has a relevant scientific background. Prospective clinical trials looking at this issue will be welcome.", "responses": [] } ]	1	https://f1000research.com/articles/5-175
https://f1000research.com/articles/5-169/v1	12 Feb 16	{ "type": "Review", "title": "Neural repair in the adult brain", "authors": [ "Sebastian Jessberger" ], "abstract": "Acute or chronic injury to the adult brain often results in substantial loss of neural tissue and subsequent permanent functional impairment. Over the last two decades, a number of approaches have been developed to harness the regenerative potential of neural stem cells and the existing fate plasticity of neural cells in the nervous system to prevent tissue loss or to enhance structural and functional regeneration upon injury. Here, we review recent advances of stem cell-associated neural repair in the adult brain, discuss current challenges and limitations, and suggest potential directions to foster the translation of experimental stem cell therapies into the clinic.", "keywords": [ "Neural repair", "brain injury", "stem cell", "Neural stem cell", "neurogenesis" ], "content": "Introduction\n\nSimilar to the rest of the body, the brain is constantly at risk of damage through either acute or chronic injury. The long-standing assumption has been that the capacity for regeneration is strongly limited in the adult mammalian brain compared with other tissues such as the skin, liver, or intestines. In line with this, the mammalian brain is not able to simply regrow lost structures that are damaged during deleterious events such as ischemic stroke or traumatic brain injury. However, there is substantial functional restoration with acute or chronic injury because of the ability of surviving neural structures to take over at least partially the previous functions of lost tissues. This becomes clear, for example, with patients who have left-hemispheric strokes and may initially suffer from motor or sensory aphasia: with extensive training and rehabilitation, a substantial number of patients regain their ability to speak and communicate1. Similarly, the brain can compensate functionally for massive loss of neural tissue before the consequences become apparent2. For example, it is considered that more than 80% of all dopaminergic neurons in the substantia nigra are lost before Parkinsonian symptoms appear3. Thus, the restorative potential of the adult mammalian brain to repair itself—at least functionally—certainly exists.\n\nHowever, at the same time, these endogenous repair mechanisms have clear limitations, leaving a substantial percentage of patients with acute or chronic injury of the adult brain with permanent functional deficits. Thus, novel strategies need to be developed to ameliorate the course of degenerative or traumatic brain diseases. Substantial efforts have been made to either recruit or enhance endogenous repair mechanisms or to ameliorate brain function in the disease context by providing exogenous cells using transplantation4,5. Here, we focus exclusively on current approaches and ideas for how endogenous neural stem cells (NSCs) or other neural cells may be used to enhance brain repair.\n\n\nNeurogenic permissiveness in the adult brain\n\nAlready in the mid-1960s, first reports suggested that the generation of neurons in the mammalian brain is not limited to embryonic or early postnatal periods but that the adult brain retains the capacity to generate new neurons6–8. These findings were met with large skepticism because they challenged a long-standing dogma in the neurosciences stating that no new neurons may be born after the end of embryonic and early postnatal development9,10. It took another 30 years and the advent of novel techniques to unequivocally identify newborn neurons in the adult brain before the process of lifelong neurogenesis in the mammalian brain became broadly accepted11,12. However, the generation of new neurons is not widespread but appears to be restricted to distinct areas of the adult brain. One of those regions is the hippocampus, a key brain structure that, simplified, serves to regulate the sorting of certain experiences into long- and short-term memory and that has been identified as a neurogenic area permitting for the lifelong addition of dentate gyrus (DG) granule cells13,14.\n\nThe finding that NSCs persist even in the adult brain spurred tremendous efforts with the aim to recruit endogenous NSCs for enhanced brain repair upon injury. In addition, the fact that new neurons that are born throughout life with the possibility to functionally integrate into pre-existing circuitries gave rise to new hope that restoration of neural circuits via transplantation approaches using exogenous NSCs or other neural cells may be feasible in principle. Here, we review multiple facets of how stem cell-associated processes may be harnessed for future regenerative approaches. Furthermore, we discuss how characterizing the neurogenic process in the adult brain may help to improve our understanding of disease etiology and progression and how this understanding may help to develop novel strategies to treat diseases of the adult brain.\n\n\nTargeting endogenous neurogenesis for brain repair\n\nNSCs generate new neurons in discrete regions of the adult brain15. In the rodent brain, two main neurogenic areas have been identified. One of those is the subventricular zone (SVZ) lining the lateral ventricles where NSCs give rise to newborn cells that migrate along the rostral migratory stream toward the olfactory bulb (OB), where they differentiate into different types of olfactory neurons16,17. Whereas SVZ/OB neurogenesis is very substantial in the rodent brain, the neurogenic activity of the SVZ seems extremely reduced or absent in the human brain18,19. This is in contrast to the second main neurogenic area: the hippocampal DG, where NSCs give rise throughout life to DG granule cells20. Multiple lines of evidence suggest that also in the human hippocampus a substantial number of neurons are born throughout life and that as a result a substantial part of the DG granule cell population is generated during postnatal life21,22.\n\nAt the top of the neurogenic lineage stand largely quiescent NSCs, called type-B cells in the SVZ and type-1 or radial glia-like NSCs in the DG, that have a number of astrocytic properties (such as expression of astroglial markers and vascular end-feet)16,23,24. Upon activation, through extrinsic and intrinsic signals, radial glia-like NSCs enter the cell cycle and give rise to more proliferative type-C cells (SVZ) or type-2 cells (DG) generating neuroblasts that eventually differentiate into newborn neurons and integrate into the DG or OB circuits over the course of several weeks25–30. Notably, the levels of neurogenesis in the adult brain are dynamically regulated with a number of positive (e.g., physical activity, learning, and environmental enrichment) and negative (e.g., stress, aging, and inflammation) regulators through a number of intrinsic and extrinsic factors13,31–40. Furthermore, it has been shown that hippocampal neurogenesis is substantially altered in a number of animal disease models13. For example, neurogenesis is reduced in animal models of major depression but enhanced upon treatment with certain antidepressants such as selective serotonin reuptake inhibitors (SSRIs)41–44. Strikingly, adult hippocampal neurogenesis seems to be required for at least some aspects of the antidepressant efficacy of SSRIs in animal models of depression45. These effects may not strictly qualify as “neural repair”, but it is reasonable to consider endogenous neurogenesis, in this case enhanced through antidepressants, as support of the improperly functioning brain to rebuild its correct connectivity.\n\nAnother example where altered neurogenesis in the adult DG may contribute to the disease process is temporal lobe epilepsy (TLE). In animal models of TLE, substantial changes in the levels of neurogenesis (acutely: enhanced; chronically: reduced) and strongly abnormal modes of neuronal integration (i.e. aberrant migration and ectopic synapse formation) have been described, suggesting that seizure-induced neurogenesis may contribute to the process of epileptogenesis and TLE-associated co-morbidities such as cognitive impairment46–52. However, there is also evidence that altered neurogenesis in animal models of TLE may rather represent an attempt of the injured brain to repair itself by balancing excess excitation that occurs in animal models of TLE53. Be that as it may, targeting neurogenesis either to prevent abnormal and ectopic neurogenesis or to foster regenerative neurogenesis in the context of TLE may reduce the development of seizures or reduce co-morbidities associated with later stages of TLE such as hippocampus-dependent cognitive decline54.\n\nFurthermore, a large number of chronic neurodegenerative diseases, such as Alzheimer’s disease, have been associated with reduced neurogenesis that may participate in the progression of observed behavioral phenotypes of these diseases13. Thus, future approaches will aim to enhance neurogenesis with the goal of either partially stopping disease progression or ameliorating secondary cognitive symptoms.\n\nWithout any doubt, the basic understanding of the function of adult neurogenesis needs to be characterized in much more detail to understand its potential role in disease processes. Newborn neurons are involved in a number of learning tasks and cognitive processes55–59. In addition, there is now ample evidence that hippocampal neurogenesis is also associated with emotional control60,61. However, it is still rather unclear when and how new neurons fulfill their action, even though accumulating data suggest that the period of heightened excitability may be critical for the effects of adult-born neurons on circuit activity27,28,62–64. Notably, it is currently believed not just that the purpose of hippocampal neurogenesis is simply to replace other neurons but that the key function of neurogenesis may be to provide young and excitable new neurons65. This may be in contrast to lifelong neurogenesis in the SVZ/OB, where, in rodents, the continuous generation of new neurons is also critically involved in proper tissue homeostasis66. Despite recent progress67,68, we still miss mechanistic data explaining the role of physiological neurogenesis that may be important to guide future experiments with the aim to harness the endogenous stem cell-associated potential for neural repair.\n\nOverall, the role for endogenous SVZ/OB neurogenesis in the context of human neural repair is less clear given the strong evidence that the neurogenic niche in the human SVZ underlies very substantial changes during the early postnatal periods that may not support lifelong neurogenesis19,69. Furthermore, novel technological approaches (such as C14-based birth dating of neurons) suggest that no or only extremely few new neurons are integrated into the human OB during adulthood18. In addition, ischemic stroke (that is sufficient to trigger at least transient neurogenesis in the rodent striatum) apparently does not lead to substantial formation of new neurons in the human cortex70–72. However, these findings clearly do not rule out the potential for SVZ-associated neurogenesis also in the human brain. In fact, a recent study showed that, despite the virtual absence of neurogenesis in the human OB, substantial numbers of newborn neurons could be detected in the human striatum that become substantially depleted in patients with Huntington’s disease, suggesting that neurogenesis outside the DG and SVZ/OB may be involved in human disease73. However, it appears that newborn striatal neurons are generated by local neurogenic astroglial cells and are not derived by NSCs residing in the SVZ73–75. Future studies will have to aim to identify the molecular and cellular details of striatal neurogenesis in rodents and humans.\n\n\nNeural stem cell-based glial repair\n\nApart from approaches aiming to enhance endogenous neurogenesis for neuronal repair in the context of acute or chronic disease, the fate potential of endogenous NSCs also permits for targeting NSCs to support glial cell replacement and subsequent neural repair. In the rodent SVZ, it has been shown, for example, that demyelination leads to enhanced NSC-derived generation of oligodendrocytes that may help to remyelinate the injured brain upon lesion76,77. Similarly, induced generation of oligodendrocytes (that are not generated by DG NSCs under normal conditions) may represent an approach to induce remyelination of the DG circuitry for several demyelinating diseases such as multiple sclerosis or epilepsy78–81. However, potential therapeutic strategies aiming to use endogenous NSCs for glial repair are currently only beginning to be developed and additional evidence for their efficacy to improve brain function needs to be generated in rodent models of human disease.\n\n\nInducing neurogenesis outside the neurogenic niches\n\nPhysiological neurogenesis from NSCs may be extremely restricted in the human brain—potentially exclusively to the DG under physiological conditions. However, recent evidence has shown that neural cells that are non-neurogenic under normal conditions may be amenable to exogenous reprogramming cues, allowing them to generate neuronal cells in vivo. This hypothesis was initially based on the fact that NSCs share many molecular and cellular features with classical astrocytes that are found throughout the brain parenchyma16,82,83. Indeed, there is now compelling evidence that providing appropriate transcriptional cues is sufficient to induce neurogenesis throughout the cortex and other regions of the central nervous system84–93.\n\nAt this time, the main target population to ectopically induce the generation of newborn neurons are astrocytes. However, there is also evidence that other glial cells such as oligodendrocytes, oligodendrocyte precursor cells, or pericytes may be targeted to induce neurogenesis throughout the brain93–98. Apart from testing different cellular populations in the injured brain that may be used to generate new neurons, the generation of neuronal subtypes is a key interest in the field with the idea to replace the exact neuronal subtype that may be preferentially lost in certain diseases (e.g., dopaminergic neurons in the context of Parkinson’s disease)97,99. These experiments are guided by pioneering work studying the mechanisms controlling brain development using cocktails or sequential overexpression of subtype-specific transcription factors. Furthermore, current studies attempt to translate data that were generated by using in vitro fate specification approaches into the in vivo situation97,100. Thus, inducing neurogenesis with high spatial control in injured brain areas may represent a promising approach for targeted brain repair.\n\n\nKey challenges and future directions\n\nThe finding that NSCs persist in the brain throughout life has been the starting point for novel approaches to enhance brain repair (Figure 1). The applications for targeting NSCs are manifold and range from their potential involvement in the disease process (e.g., major depression) to their ability to generate new neuronal and glial cells (e.g., neurodegenerative diseases such as Alzheimer’s disease). In addition, approaches to induce neurogenesis outside physiological neurogenic niches may be of translational value5,101. However, we are still only beginning to understand what it takes for new neurons to truly make a functional impact on the injured brain. Key to improving these approaches will be to identify the mechanisms that regulate meaningful and proper integration into existing circuitries. This may be more feasible for some diseases where neurons may rather fulfill the function of providing neurotransmitters such as dopamine, but potentially more challenging and further away from clinical applications if diffuse circuitries or complete brain areas are impaired or destroyed.\n\nFuture experiments will relate basic research findings obtained mostly in laboratory animals to the analyses of human disease and eventually to the therapeutic targeting of endogenous neural stem cells, the improved use of transplantation-based cell replacement strategies, or the reprogramming of other neural cells with the aim to enhance the potential for repair of the adult human brain. The road toward translation may lead from understanding physiologic and disease-associated neurogenesis in humans and an improved understanding of the molecular and cellular mechanisms underlying the neurogenic process toward novel approaches to study human diseases in the dish and mouse models. Finally, the application of this knowledge may lead to enhanced recruitment of endogenous stem cells or improved functionality of transplants and reprogramming-based approaches for neural repair. hESC, human embryonic stem cell; iPSC, induced pluripotent stem cell.\n\nApart from increasing our understanding of the potential of endogenous NSCs or other neural cells for brain repair, the detailed molecular and cellular characterization of these processes also may be helpful to guide and improve current attempts to ameliorate brain function upon injury using exogenous transplantation of NSCs or other neural cells4,101. The key questions, such as neuronal differentiation, control of growth, and proper neuronal integration, are shared between these two strategies (NSC activation versus transplantation-based approaches) to target endogenous neural cells and to support brain repair with exogenous cells. In addition, it is foreseeable that ongoing studies aiming to understand disease processes using human embryonic stem cells or induced pluripotent stem cell-based approaches not only will improve our understanding of disease mechanisms but also may guide future strategies to enhance endogenous neural repair.\n\n\nAbbreviations\n\nDG, dentate gyrus; NSC, neural stem cell; OB, olfactory bulb; SSRI, selective serotonin reuptake inhibitor; SVZ, subventricular zone; TLE, temporal lobe epilepsy.", "appendix": "Competing interests\n\n\n\nThe author declares that he has no competing interests.\n\n\nGrant information\n\nThe authors declares that no grants were involved in supporting this work.\n\n\nReferences\n\nPedersen PM, Jørgensen HS, Nakayama H, et al.: Aphasia in acute stroke: incidence, determinants, and recovery. Ann Neurol. 1995; 38(4): 659–666. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "12430", "date": "12 Feb 2016", "name": "Benedikt Berninger", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12431", "date": "12 Feb 2016", "name": "Chun-Li Zhang", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12432", "date": "12 Feb 2016", "name": "Hongjun Song", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-169
https://f1000research.com/articles/5-168/v1	12 Feb 16	{ "type": "Review", "title": "Diagnosis, prognosis, and management of cryptogenic stroke", "authors": [ "Cen Zhang", "Scott Kasner", "Scott Kasner" ], "abstract": "Despite many advances in our understanding of ischemic stroke, cryptogenic strokes (those that do not have a determined etiology) remain a diagnostic and therapeutic challenge. Previous classification approaches to cryptogenic stroke have led to inconsistent definitions, and evidence to determine optimal treatment is scarce. These limitations have prompted international efforts to redefine cryptogenic strokes, leading to more rigorous diagnostic criteria, outcome studies, and new clinical trials. Improvement in our ability to detect paroxysmal atrial fibrillation in patients with cryptogenic stroke has strengthened the idea that these strokes are embolic in nature. Further, better understanding of acute biomarkers has helped to identify otherwise occult mechanisms. Together, these strategies will inform long-term outcomes and shape management.", "keywords": [ "Cryptogenic Stroke", "diagnosis of cryptogenic stroke", "prognosis of cryptogenic stroke", "secondary stroke prevention", "Management of cryptogenic stroke" ], "content": "Introduction\n\nCryptogenic stroke refers to stroke of unknown etiology and accounts for approximately 15–40% of all ischemic strokes1,2. Though cryptogenic stroke seems common, the term lacks specificity and leads to great variability among studies. Moreover, there are no randomized controlled trials to guide long-term treatment. In this review, we will discuss current and emerging diagnostic criteria for cryptogenic stroke, long-term outcomes, and therapeutic options.\n\n\nDiagnosis\n\nThe diagnosis of cryptogenic stroke has traditionally been based on the exclusion of other well-established causes of stroke3. Three classification systems have frequently been employed to define subtypes or mechanisms of ischemic stroke: the Trial of Org 10172 in Acute Stroke Treatment (TOAST) system, the Causative Classification of Stroke System (CCS), and the Atherosclerosis, Small vessel disease, Cardiac causes, Other, and Dissection (ASCOD) scheme4–6. None formally define cryptogenic stroke. TOAST includes a category of “stroke of undetermined etiology”, which includes strokes of unknown source despite an extensive evaluation, as well as those with incomplete evaluation and those with more than one identified etiology. CCS offers a category of “undetermined stroke”, which similarly includes subcategories of unknown-cryptogenic embolism, unclassified, and incomplete evaluation. ASCOD has no specific category for stroke of unknown cause but includes them in the category of “other”. While all three systems differ in how unknown or cryptogenic stroke is defined, with different inter-rater reliability, they all require exclusion of other well-established singular etiologies. Notably, these systems do not mandate a minimum set of diagnostic tests, and as a result they will classify several distinct groups as cryptogenic, including patients who had a very extensive evaluation that proved normal, and those with very limited or even no testing that also was unrevealing. Arguably, a thorough evaluation requires brain imaging, with computed tomography (CT) or magnetic resonance imaging (MRI), neurovascular imaging with CT angiography (CTA), MR angiography (MRA) or cervical carotid duplex and transcranial Doppler, cardiac evaluation with echocardiography, and, in select patients, rapid plasmin reagin (RPR), erythrocyte sedimentation rate (ESR), hypercoagulable testing, genetic analysis, or other tests for atypical causes. A broader consensus is required to define the characteristics and criteria for a diagnosis of cryptogenic stroke.\n\nRecent evidence suggesting cryptogenic stroke is likely due to embolic sources has altered these familiar but vague definitions, leading to a new and more rigorously defined term, embolic strokes of undetermined source (ESUS). Introduced by the Cryptogenic Stroke/ESUS International Working Group, this term offers a way to define cryptogenic stroke based on established criteria, rather than due to the lack of an explanation. Diagnostic criteria for ESUS include brain CT or MRI to demonstrate non-lacunar stroke, extracranial and intracranial imaging to exclude ≥50% proximal stenosis, and electrocardiography, echocardiography, and cardiac rhythm monitoring for ≥24 hours to exclude cardioembolic sources7. The ESUS definition likely remains highly heterogeneous, including cardiac abnormalities of uncertain risk (e.g. covert paroxysmal atrial fibrillation [AF], mitral annular calcification, aortic valve disease, or atrial pathology), arteriogenic embolism (e.g. from a nonstenotic ulcerated plaque), paradoxical embolism (e.g. patent foramen ovale or pulmonary arteriovenous malformation), and unknown prothrombotic disorders (e.g. occult malignancy), but provides a useful construct for clinical and research purposes.\n\nBetter understanding of the pathophysiology of cryptogenic stroke may also improve its diagnosis and characterization. For example, acute blood biomarkers, including brain natriuretic peptide (BNP), N-terminal proBNP (NT-proBNP), and D-dimer, have emerged as potential aids in determining the underlying etiology of cryptogenic stroke. In a recent meta-analysis of 2834 patients, levels of BNP and NT-proBNP were significantly elevated in patients with cardioembolic stroke, independent of other clinical factors8. In addition, a post-hoc analysis of a subset of participants in the Warfarin vs. Aspirin for Recurrent Stroke Study (WARSS) demonstrated no difference between aspirin and warfarin on the risk of stroke or vascular death when NT-proBNP level was ≤750 pg/mL (hazard ratio [HR] 1.21, p=0.243), but warfarin reduced the risk compared to aspirin when NT-proBNP levels were >750 pg/mL (HR 0.30, p=0.021)9. These studies suggest that acutely elevated levels of BNP in patients with cryptogenic stroke may harbor an underlying or occult cardioembolic mechanism.\n\nSimilarly, acutely elevated D-dimer levels after a stroke may implicate a hypercoagulable state secondary to an occult malignancy. A study by Schwarzbach et al. comparing 140 patients with cancer and ischemic stroke to 140 age- and sex-matched control patients with stroke alone demonstrated that cancer was associated with a higher prevalence of unidentified strokes (48% vs. 27%, p<0.001) as well as higher levels of D-dimer (6.15 µg/mL vs. 1.39 µg/mL, p<0.001)10. An analysis by Kim et al. similarly showed higher levels of D-dimer in patients with cancer and cryptogenic stroke compared to those with cryptogenic stroke without cancer as well as a control group of patients with cancer without stroke11.\n\n\nPrognosis\n\nThe prognosis of cryptogenic stroke varies, which likely reflects the heterogeneity of the definition as well as the shortage of studies. Several new studies examining long-term outcomes among stroke subtypes, including ESUS, provide comparative data. A retrospective cohort analysis at the Helsinki University Hospital examined recurrent stroke and death risk in a subset of patients referred to as having had an undetermined stroke with an embolic pattern (USEP)12. These patients had embolic lesions on neuroimaging without having completed a full diagnostic evaluation and were further classified into whether or not they met the cryptogenic stroke/ESUS criteria. Among 540 patients with ischemic stroke 23.5% were classified as USEP, and within this group 36.2%, or 8.5% of all patients, met criteria for ESUS. At 21 months, USEP was associated with a higher risk of recurrent stroke compared to both noncardioembolic (HR 2.36, p=0.046) and cardioembolic strokes with known source (HR 1.83, p=0.028). Among the USEP subgroup, there was no difference in risk of recurrent stroke between those who met ESUS criteria versus those who did not.\n\nMore recently, Ntaios et al. described the long-term outcomes of ESUS patients in the Athens Stroke Registry13. This retrospective analysis included 2731 patients with first ischemic strokes between 1992 and 2011, followed for a mean of 31 months; 10% were diagnosed as ESUS using the cryptogenic stroke/ESUS International Working Group criteria. In this population, the cumulative probability of stroke recurrence in ESUS was similar to cardioembolic strokes (29% vs. 27%) but higher than all other types of noncardioembolic stroke, including large artery atherosclerosis (13%) and lacunar strokes (13%). Notably, there was a higher percentage of ESUS patients with a favorable functional outcome, defined as modified Rankin scale (mRS) ≤2 (62.5%), compared to patients with cardioembolic strokes (32.2%).\n\n\nManagement\n\nThe major challenge in managing cryptogenic stroke is secondary stroke prevention of cryptogenic strokes, specifically in choosing antithrombotic therapy. The use of oral anticoagulation for secondary prevention of cardioembolic strokes is well established, and cryptogenic strokes are being recognized as sharing many features with cardioembolic strokes. However, there are currently no well-founded guidelines for optimal long-term treatment. According to the American Heart Association/American Stroke Association and the American College of Chest Physicians, antiplatelet agents are preferred for noncardioembolic ischemic strokes. A global survey of hospitals in 48 countries found that the vast majority (94%) routinely prescribed antiplatelet therapy for secondary prevention of cryptogenic stroke14, yet there is growing evidence that cryptogenic stroke patients may benefit from anticoagulation. To date, the only randomized trial data comparing the efficacy of anticoagulation to antiplatelet therapy in cryptogenic stroke are derived from post-hoc analyses of the WARSS trial. While the primary analysis of WARSS showed no significant advantage of warfarin compared to aspirin in secondary prevention of noncardioembolic strokes, the subgroup analysis suggested that warfarin was associated with one-third fewer recurrent strokes than aspirin in cryptogenic stroke patients with an embolic appearance, though the result did not reach statistical significance15.\n\nDespite the absence of large, randomized controlled trials, emerging data linking cardiac abnormalities to cryptogenic strokes have shifted management increasingly in favor of anticoagulation. Recent studies show that using prolonged cardiac monitoring devices provides better detection of paroxysmal AF in patients with cryptogenic stroke16. The Cryptogenic Stroke and Underlying AF (CRYSTAL-AF) trial demonstrated that the use of an implantable cardiac monitor increased the rate of AF detection significantly compared to standard monitoring at 6 months (8.9% vs. 1.4%, p<0.001)17. Similarly, the 30-Day Cardiac Event Monitor Belt for Recording AF After a Cerebral Ischemic Event (EMBRACE) trial showed that use of a 30-day loop recorder increases the yield of AF detection in patients diagnosed with cryptogenic stroke (16.1% vs. 3.2%, p<0.001)18. While neither of these studies claimed a causal link between paroxysmal AF and cryptogenic stroke, there was a significantly higher rate of subsequent oral anticoagulation use in the group who underwent prolonged monitoring. In CRYSTAL-AF, anticoagulant use was observed in 10.1% of the intervention group, compared to 4.6% of the control group at 6 months (p=0.04), and 14.7% versus 6% at 12 months (p=0.007). In EMBRACE, anticoagulant use at 90 days was 18.6% in the intervention group compared to 11.1% of the control group (p=0.01). In addition, there was a higher rate of conversion from antiplatelet to anticoagulation in the EMBRACE intervention group compared to the control group (13.6% versus 4.7%, p<0.001). The Asymptomatic Atrial Fibrillation and Stroke Evaluation in Pacemaker Patients and the Atrial Fibrillation Reduction Atrial Pacing Trial (ASSERT) demonstrated that in patients without a prior history of AF, detection of subclinical atrial tachyarrhythmia (AT) lasting at least 6 minutes by pacemakers or implantable cardioverter-defibrillators was correlated with a 2.5-fold increased risk of ischemic stroke (p=0.008)19. Taken together, these studies imply that detection of occult AF in cryptogenic stroke may warrant treatment with anticoagulation. A standard approach in our practice is to monitor patients in whom cardioembolic source is strongly suspected for 30 days using a mobile cardiac outpatient telemetry unit (MCOT). Patients typically remain on single antiplatelet therapy unless AF is detected.\n\nA major area of ongoing uncertainty relates to the minimal duration of AF needed to increase the risk of ischemic stroke and the total burden needed to warrant treatment with anticoagulation19,20. While the recent advances in technology allow for detection of AF episodes even less than 30 seconds, there are no reliable data demonstrating a clear role for anticoagulation in such circumstances. Thus, how to treat patients in whom short-duration AF is detected after device implantation remains unclear, and is the focus of ongoing trials including Apixaban for the Reduction of Thrombo-Embolism in Patients with Device-Detected Sub-Clinical Atrial Fibrillation (ARTESiA), a prospective study to assess whether anticoagulation reduces risk of stroke and systemic embolism in patients with device-detected subclinical AF21.\n\nFurther, the detection of paroxysmal AF does not necessarily provide an etiologic mechanism for cryptogenic stroke. A follow-up study by the ASSERT investigators questioned the causality of subclinical AT and ischemic stroke, noting that arrhythmia detection was temporally not related to the index stroke event20. These findings suggest that paroxysmal AF may be a risk factor or marker for other comorbidities that increase the risk of stroke, rather than the sole or primary etiology. In contrast, Turakhia et al. recently established a temporal relationship through a case-crossover analysis of patients with implanted cardiac devices who developed AF and stroke22. Within a subset of patients in whom 120 days of monitoring was available, an episode of AF that was at least 5.5 hours in a day increased the short-term risk of stroke by 4- to 5-fold in the 5 to 10 days after the AF event and decreased over time.\n\nFinally, the role of novel oral anticoagulants in cryptogenic stroke has yet to be determined. Their superior efficacy and improved safety profile compared to warfarin have prompted interest in the prevention of strokes presumably due to embolism, beyond those attributed to nonvalvular AF. Two recently launched randomized controlled trials, Rivaroxaban Versus Aspirin in Secondary Prevention of Stroke and Prevention of Systemic Embolism in Patients With Recent Embolic Stroke of Undetermined Source (NAVIGATE ESUS) and Dabigatran Etexilate for Secondary Stroke Prevention in Patients With Embolic Stroke of Undetermined Source (RE-SPECT ESUS), will assess the efficacy of rivaroxaban and dabigatran, respectively, compared to aspirin in secondary prevention of cryptogenic stroke specifically in ESUS patients.\n\n\nConclusion\n\nOur understanding of cryptogenic strokes has advanced with better technology and ongoing efforts to redefine this category. Newer cardiac monitoring devices continue to improve our ability to detect subclinical AF. Studies are underway to help us understand how to interpret these findings and which treatments improve clinical outcomes. Shifting from cryptogenic stroke to ESUS may create a stronger framework, within which we can provide focused evaluations and uniform criteria for future clinical trials. And, lastly, the completion of NAVIGATE ESUS and RE-SPECT ESUS may provide two new alternatives to our armamentarium of therapeutics.", "appendix": "Competing interests\n\n\n\nCen Zhang declares that she has no competing interests. Scott Kasner is a consultant to Bayer, Johnson & Johnson, Bristol Myers Squibb, Boehringer Ingelheim, Daiichi Sankyo, Medtronic, and Biotechnology, Inc.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nLeys D, Bandu L, Hénon H, et al.: Clinical outcome in 287 consecutive young adults (15 to 45 years) with ischemic stroke. Neurology. 2002; 59(1): 26–33. PubMed Abstract \| Publisher Full Text\n\nKolominsky-Rabas PL, Weber M, Gefeller O, et al.: Epidemiology of ischemic stroke subtypes according to TOAST criteria: incidence, recurrence, and long-term survival in ischemic stroke subtypes: a population-based study. Stroke. 2001; 32(12): 2735–40. PubMed Abstract \| Publisher Full Text\n\nMohr JP: Cryptogenic stroke. N Engl J Med. 1988; 318(18): 1197–8. PubMed Abstract \| Publisher Full Text\n\nAdams HP Jr, Woolson RF, Clarke WR, et al.: Design of the Trial of Org 10172 in Acute Stroke Treatment (TOAST). Control Clin Trials. 1997; 18(4): 358–77. PubMed Abstract \| Publisher Full Text\n\nAy H, Benner T, Arsava EM, et al.: A computerized algorithm for etiologic classification of ischemic stroke: the Causative Classification of Stroke System. Stroke. 2007; 38(11): 2979–84. PubMed Abstract \| Publisher Full Text\n\nAmarenco P, Bogousslavsky J, Caplan LR, et al.: The ASCOD phenotyping of ischemic stroke (Updated ASCO Phenotyping). Cerebrovasc Dis. 2013; 36(1): 1–5. PubMed Abstract \| Publisher Full Text\n\nHart RG, Diener HC, Coutts SB, et al.: Embolic strokes of undetermined source: the case for a new clinical construct. Lancet Neurol. 2014; 13(4): 429–38. PubMed Abstract \| Publisher Full Text\n\nLlombart V, Antolin-Fontes A, Bustamante A, et al.: B-type natriuretic peptides help in cardioembolic stroke diagnosis: pooled data meta-analysis. Stroke. 2015; 46(5): 1187–95. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nLongstreth WT Jr, Kronmal RA, Thompson JL, et al.: Amino terminal pro-B-type natriuretic peptide, secondary stroke prevention, and choice of antithrombotic therapy. Stroke. 2013; 44(3): 714–9. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nSchwarzbach CJ, Schaefer A, Ebert A, et al.: Stroke and cancer: the importance of cancer-associated hypercoagulation as a possible stroke etiology. Stroke. 2012; 43(11): 3029–34. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nKim SJ, Park JH, Lee MJ, et al.: Clues to occult cancer in patients with ischemic stroke. PLoS One. 2012; 7(9): e44959. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nPutaala J, Nieminen T, Haapaniemi E, et al.: Undetermined stroke with an embolic pattern--a common phenotype with high early recurrence risk. Ann Med. 2015; 47(5): 406–13. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nNtaios G, Papavasileiou V, Milionis H, et al.: Embolic Strokes of Undetermined Source in the Athens Stroke Registry: An Outcome Analysis. Stroke. 2015; 46(8): 2087–93. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nGiruparajah M, Bosch J, Vanassche T, et al.: Global survey of the diagnostic evaluation and management of cryptogenic ischemic stroke. Int J Stroke. 2015; 10(7): 1031–6. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nSacco RL, Prabhakaran S, Thompson JL, et al.: Comparison of warfarin versus aspirin for the prevention of recurrent stroke or death: subgroup analyses from the Warfarin-Aspirin Recurrent Stroke Study. Cerebrovasc Dis. 2006; 22(1): 4–12. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nChoe WC, Passman RS, Brachmann J, et al.: A Comparison of Atrial Fibrillation Monitoring Strategies After Cryptogenic Stroke (from the Cryptogenic Stroke and Underlying AF Trial). Am J Cardiol. 2015; 116(6): 889–93. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nSanna T, Diener HC, Passman RS, et al.: Cryptogenic stroke and underlying atrial fibrillation. N Engl J Med. 2014; 370(26): 2478–86. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nGladstone DJ, Spring M, Dorian P, et al.: Atrial fibrillation in patients with cryptogenic stroke. N Engl J Med. 2014; 370(26): 2467–77. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nHealey JS, Connolly SJ, Gold MR, et al.: Subclinical atrial fibrillation and the risk of stroke. N Engl J Med. 2012; 366(2): 120–9. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nBrambatti M, Connolly SJ, Gold MR, et al.: Temporal relationship between subclinical atrial fibrillation and embolic events. Circulation. 2014; 129(21): 2094–9. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nHealey JS: Apixaban for the Reduction of Thrombo-Embolism in Patients with Device-Detected Sub-Clinical Atrial Fibrillation. In: ClinicalTrials.gov. Bethesda (MD): National Library of Medicine. 2015. Reference Source\n\nTurakhia MP, Ziegler PD, Schmitt SK, et al.: Atrial Fibrillation Burden and Short-Term Risk of Stroke: Case-Crossover Analysis of Continuously Recorded Heart Rhythm From Cardiac Electronic Implanted Devices. Circ Arrhythm Electrophysiol. 2015; 8(5): 1040–7. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation" }	[ { "id": "12422", "date": "12 Feb 2016", "name": "Vincent Thijs", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12421", "date": "12 Feb 2016", "name": "Brian Silver", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12427", "date": "12 Feb 2016", "name": "Konstantinos Vemmos", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-168
https://f1000research.com/articles/5-167/v1	12 Feb 16	{ "type": "Opinion Article", "title": "Understanding the nature of health: New perspectives for medicine and public health. Improved wellbeing at lower costs", "authors": [ "Johannes Bircher", "Eckhart G. Hahn", "Eckhart G. Hahn" ], "abstract": "Background: Current dilemmas of health care systems call for a new look at the nature of health. This is offered by the Meikirch model. We explore its hypothetical benefit for the future of medicine and public health.Meikirch model: It states: “Health is a dynamic state of wellbeing emergent from conducive interactions between individuals’ potentials, life’s demands, and social and environmental determinants.” “Throughout the life course health results when an individuals’ biologically given potential (BGP) and his or her personally acquired potential (PAP), interacting with social and environmental determinants, satisfactorily respond to the demands of life.”Methods: We explored the Meikirch model’s possible applications for personal and public health care.Results: The PAP of each individual is the most modifiable component of the model. It responds to constructive social interactions and to personal growth. If an individual’s PAP is nurtured to develop further, it likely will contribute much more to health than without fostering. It may also compensate for losses of the BGP. An ensuing new culture of health may markedly improve health in the society. The rising costs of health care presumably are due in part to the tragedy of the commons and to moral hazard. Health as a complex adaptive system offers new possibilities for patient care, particularly for general practitioners.Discussion: Analysis of health systems by the Meikirch model reveals that in many areas more can be done to improve people’s health and to reduce health care costs than is done today. The Meikirch model appears promising for individual and public health in low and high income countries. Emphasizing health instead of disease the Meikirch model reinforces article 12 of the International Covenant on Economic, Social and Cultural Rights of the United Nations – that abandons the WHO definition - and thereby may contribute to its reinterpretation.", "keywords": [ "Definition of health", "Meikirch model", "innovative health policies", "potentials for health", "health as a complex adaptive system", "responsibility for health", "social aspects of health", "culture of health", "health economy" ], "content": "Introduction\n\nOver the past century, biological and medical sciences have accumulated an enormous amount of knowledge and expertise. Today, the understanding of biological processes ranging from genetic mechanisms to organ function of complex living systems is huge. Much of it has been made available to individual and public health in order to prevent and treat diseased humans. As a result the current state of personal health can be seen as a major accomplishment. Life expectancy in high income countries has approximately doubled in the past 130 years (e.g., in Switzerland from 40.5 to 82.5 years)1. Nevertheless, non-communicable diseases remain a major target of the World Health Organization (WHO)2. The years lived with disabilities increases progressively and multimorbidity augments the demands on health care systems3.\n\nOne way to further improve the current situation may be to clarify what health truly is, i.e. to offer a valid concept of health that can be applied to the care of individuals and to public health. For this purpose a new definition of health, the Meikirch model, was developed and published in 20144,5. As a result there are now firm theoretical indications about what might be achieved once the model is implemented. This paper has the purpose of initially briefly explaining the Meikirch model. Thereafter, investigations of its possible consequences focus first on the special role of the personality of each individual for his or her own health and shows how it may be supported further. Subsequently, the social and environmental determinants of health are reviewed in the light of the Meikirch model. E.g. development of a culture of health may substantially improve the health of whole populations. The rise in health care costs may be reversed because the Meikirch model counterbalances the tragedy of the commons. Health explained as a complex adaptive system offers new opportunities, particularly for general practitioners and rehabilitation institutions. We feel that putting the Meikirch model into practice could result in significant further world-wide improvements in health with decreased rather than increased costs.\n\n\nMeikirch model (Figure 1)\n\nThe wording of the definition of health given by the Meikirch model is as follows4,5: “Health is a dynamic state of well-being emergent from conducive interactions between an individual’s potentials, life’s demands, and social and environmental determinants.” “Health results throughout the life course when an individual’s potentials – and social and environmental determinants – suffice to respond satisfactorily to the demands of life. Life’s demands can be physiological, psychosocial, or environmental, and vary across individuals and contexts, but in every case unsatisfactory responses lead to disease.”\n\nWith this in mind, the Meikirch model consists of five components and ten complex interactions. These are shown in Figure 1 and Table 1, where the five components are depicted and the interactions are exhibited as double-edged arrows. The complete description of the model with its scientific background is given in the original publication. Here only a short explanation of the five components and their interactions is presented.\n\n(Abbreviations: BGP = biologically given potential and PAP = personally acquired potential).\n\nHumans, like all other biological creatures, are exposed to the demands of life (Figure 1). Their fulfilment is a condition for life6. In people, these demands are physiological, psychosocial and environmental.\n\n1. Physiological demands. Humans have to meet physiological needs that vary with time and circumstances. They present themselves in many ways related to input, output, homeostasis, work, and procreation. “Intake of oxygen, nutrients and water, excretion, fertilization, pregnancy and childbirth are key examples. Some specific characteristics differentiate humans from other higher animals, e.g., the choice of whether or not to procreate”4.\n\n2. Psychosocial demands. “Psychosocial demands relate to individuals’ personal development and social integration, including participation in the social, economic and political life. All these are interlinked. Each individual is exposed to various social determinants of health throughout the life course, with varying roles and expectations, as related to jobs, relationships, obligations to family and society, personal aspirations and political and economic contexts”4. The way in which life’s psychosocial demands present themselves and can be fulfilled depends on the specifics of the respective individual and society. The final psychosocial demand is to achieve peace with the fact that every human must die.\n\n3. Environmental demands. Environmental factors may decrease or increase the demands of life in many ways, thereby affecting personal health. Examples are availability of clean drinking water, conditions for food production, air pollution, radioactivity and safe workplaces. In addition, protection from physical, chemical, and microbiological threats and safe disposal of waste matter (recycling) is essential. Some of these are apparent immediately, while others could be latent for many years (e.g., exposure to carcinogens from tobacco smoke, pollutants or radioactivity).\n\nEach person needs resources to meet the demands of life. They have to satisfy the demands both in the present and in the long term. In order to serve the purposes of the Meikirch model the term potential was introduced to express both present and future resources. The potentials of an individual person have a double nature, parts are given and parts are acquired. Individuals always draw simultaneously on both potentials to meet life’s demands as outlined below.\n\nBiologically given potential (BGP). The BGP represents the biological basis of life. At the moment of birth it has a finite value resulting from genetic equipment, epigenetic regulation and quality of the pregnancy. This is the gift of life everybody receives. This potential diminishes naturally throughout life, reaching zero at the time of death. Every substantial social disadvantage, somatic disease, injury, or defect diminishes the BGP either transiently or permanently.\n\nPersonally acquired potential (PAP). The PAP is the sum of all physiological, mental, spiritual and social resources a person acquires during life. Presumably it starts to develop in utero while the baby hears the heart beat and the voice of the mother and feels her bodily movements. After birth, as the brain and other organs mature, this potential increases rapidly. Interactions in families, schools, and communities play a pivotal role for children and adolescents in supporting their acquisition of knowledge and skills and personal development. Even in adulthood the development of the potential continues, provided an individual cares for it. The social determinants of health have a very important enabling or inhibiting function. Integration and participation in the society is of outstanding importance for each individual. The society also provides many other helpful possibilities, such as work, food shops, health information, social security, and a health care system. Finally, the environmental determinants of health exhibit decisive enabling and threatening functions for the PAP of each individual.\n\nSocial determinants may be supportive or challenging for people’s health by modifying the demands of life and by supporting or hindering the development of their potentials. This starts in families and continues in schools and during professional formation7,8. These influences result from interacting individuals or as a result of the prevailing culture in the society. Throughout life social concerns, mutual trust, and collective efficacy support a good health outcome. Wilkinson and Pickett identified better health of people in countries with less inequality of incomes9. In many parts of the world the social determinants of health are not optimal. Poverty, difficult living and work conditions may limit the health people can achieve. “Longevity is not solely related to people’s income, but is also strongly affected by their autonomy and social participation, which are major determinants of health”8.\n\nIn 1987 the UN World Commission on Environment and Development’s report, Our Common Future, also known as the Brundtland report, noted: The \"environment\" is where we all live; and \"development\" is what we all do in attempting to improve our lot within that abode10. Factors in living and work environments may not be cared for sufficiently, e.g. global warming11. Also coal mining is an important environmental cause of diseases as are water-borne contaminants. Use of antibiotics in agriculture has contributed to human infections with resistant germs. “Adopting cleaner, more sustainable energy technologies and water sources could help promote both health and development. At the macro level, dwindling natural resources, population growth, and the effects of climate change are likely to affect global health”4.\n\nA complex adaptive system is an entity with a boundary between it and its environment, that can take up material and energy from the environment (input), release end products (output of entropy) and do work12–14. Within the system there are many different parts called agents. They continuously interact with each other in a nonlinear manner, resulting in an outcome, called emergence, that is more than the sum of the properties of these agents. In the Meikirch model the five components, including their subcomponents, are regarded as agents (Figure 1). They spontaneously and autonomously arrange themselves in such a way that the evolving products are an emergent expression of the functioning of the system as a whole. These arrangements are operational, but are not necessarily the best solution for the system. Nevertheless, in complex adaptive systems, including humans, emergence is definitely more than the sum of the properties and functions of the parts.\n\nIn a complex adaptive system there is a flow of energy. Via their inputs all living organisms take up energy from their surroundings. Humans have within themselves an additional source of energy, e.g. vitality, drive and sense of purpose15. One part of the total energy is spent to maintain the person physically and emotionally and another part to do work. The material output (excretions) contains the end products and represents entropy. The flow of energy in human beings also leads to a desire for being loved, pursuing values, and living for a purpose. Investigating the double nature of this energy flow of an individual helps to better understand her or his state of health.\n\n\nMethods\n\nFor the purpose of this investigation the Meikirch model was used as a basis to investigate selected possibilities to positively influence personal and public health as described in the Results section. In each instance two questions were asked: Does the examined situation agree with or contradict the Meikirch model? If it agrees, does the Meikirch model offer new perspectives for individual and/or public health? The deduction/induction cycles about the findings were applied repeatedly until the results appeared to be consistent.\n\n\nResults\n\nThe Meikirch model is designed to support a new, unified approach to health and disease under various conditions in diverse health systems. Some results of such an approach are delineated in the following section:\n\nEnvironmental determinants of health have impacts on the two human potentials and on the demands of life (Figure 1). Geography may dictate, for example, how and which type of food can be produced, and how housing and clothing has to be. In addition, the demands of life vary with the social determinants of health. They differ in low income and high-income countries. Demands also vary from person to person and in different cultural backgrounds. In some places women are primarily concerned with raising children, whereas men care for food production. In others the tasks are different. But role models change continuously. It often appears that not much can be done to modify the demands of life. For young individuals, therefore, it is important to choose wisely among them and, if possible, get away from life conditions that are detrimental to health. In the future, for a given cultural background, this might be reflected in appropriate educational programmes.\n\nAfter a healthy pregnancy the BGP at birth is a gift of nature and its vitality may vary from person to person. Thereafter it must be cared for. Social factors may foster or curb physical exercise, quality and quantity of food, alcohol intake, smoking, and consumption of narcotic drugs. These features are well known in pedagogy.\n\nIn earlier times epidemics have wiped out large portions of populations. Acute and chronic disease burdens threatened the BGP. Today, improved social determinants of health, e.g., public health and medical programmes, have achieved healthy lives and long life spans for a majority of the people in high income countries whereas low-income countries are still concerned with further developing their public health and medical services. Everywhere economic factors limit in one way or another what can be done for the BGP.\n\nThe PAP is presumably the most fragile and modifiable component of the Meikirch model. It is subject to strong influences through interactions with other components of the model. The PAP is a critical factor for the achievement of satisfactory or unsatisfactory responses to the demands of life. Thereby it strongly influences the realization of health or disease. Consequently, it is of greatest interest to review the factors that may promote or impede the development of this potential.\n\n1. Important factors that modify the PAP. In each individual this potential is small at birth, yet grows rapidly thereafter. From the first day children mature with the love and care of their parents19. Later, teachers play important roles. Eventually individuals have to assume responsibility for their PAP themselves. Yet, they remain affected by their social settings. Although every person continuously has to contribute him- or herself to this potential, it may be supported further by trusting, loving, respectful, and life affirming human relationships. A well-founded sense of purpose in life strengthens it also16. Alternatively it may be damaged by stress, adverse social circumstances like educational deprivation, sexual exploitation, and abuse of alcohol and narcotic drugs. Theoretically, at any moment, each individual has choices about how to handle his or her life situation. In order to choose from the existing possibilities as wisely as possible, each person develops more or less personal leadership17. Thus, the PAP of any individual is the result of complex interactions between the respective personality and his or her social settings.\n\n2. A high PAP is important for health. The PAP is critical for the choices an individual makes about how to approach the demands of life. Affinity for certain types of work, ambitions, vitality, attitudes toward rewards, etc. will influence the choices of and dedication for work. These aspects apply equally to work at home, including attention to children as well as to work outside the home, e.g., professional contributions to the society. Ideally the specifics of each work situation are matched with knowledge and skills of each worker.\n\nAttitude, culture, willingness to learn theoretically and from experience, and the possibility to grow with new challenges will have major influences on health. This is exemplified by several approaches: In his concept of salutogenesis, Antonovsky postulates that a high sense of coherence is essential for successful coping15. Comprehensibility, manageability and meaningfulness are central features of this sense of coherence. Presumably they can be learned and life managed correspondingly. Also van Spijk postulates that leading a life that makes sense results in “human great health”18. Martin Seligman found that positive psychology is closely related to health19. Flourishing persons who experience more than three positive feelings for every negative feeling are healthier and live longer than languishing persons. In the Meikirch model this may be explained by the contribution of positive emotions to the PAP. Overarching positive feelings or positive self-perception strongly influence the approach to life as shown by the following two studies. Nuns with more positive emotions at the age of about 22 years survived six years longer than nuns with more negative emotions at the same age20. “Individuals with more positive self-perceptions of aging lived 7.5 years longer than those with less positive self-perceptions”4,21. Other examples are given in Table 2. Methods to improve the PAP including its implication for health need to be investigated urgently and in depth.\n\n3. Personal health leadership. In principle every person has considerable influence on his PAP. Consequently, in as much as humans are in control of themselves, it is necessary to consider that they also have to assume personal responsibility for their health. Correspondingly, they may or may not “lead a healthy life”. This popular expression implies that the importance of leadership for personal health has been known publicly for a long time. If so, everyone might be considered as an entrepreneur of his or her own health. This is an encouraging, positive empowerment for health that may motivate people to invest in it. This idea as expressed in the Meikirch model is now being practised successfully in indigenous villages in Odisha, India22. The essence of the model was easily understood even by analphabetic persons and it changed their behaviour into a much more health supporting pattern. Until now the idea of personal responsibility for health has not received sufficient attention. This presumably is due to the fact that in the past the origin of many diseases has been unexplained and therefore diseased humans were not to be made responsible unjustly. In 2003, however, WHO has launched its campaign against non-communicable diseases23. These conditions are thought to be largely preventable by health protecting behaviour. This justifies the concept that all humans are called to assume personal leadership for their health.\n\n4. Education for a culture of health. Leadership for one's PAP might further be reinforced by introduction of a culture of health24. For this purpose all professionals of public health and individual medical care might be involved. They should teach about the Meikirch model and the relevant health conditions to students and lay persons from kindergarten up to professional maturity and old age. The principles for transformative learning worked out for health professionals may be applied equally to the general population25. For example teaching patients with type 2 diabetes improved their health26. Schools, universities, and the media should play an important role. Voices and actions of public role models might also be encouraging. In addition to the Meikirch model, easily understandable information about prevention and treatment of prevalent diseases appears to be essential for the general public. A central organization e.g., a “National Institute of Health Information” might be made available as a library and reference centre of trustworthy health information for the whole population. Once a new culture of health is established, an impressively improved health of the people most likely will be the result.\n\n5. Vaccination and levels of control of infectious diseases. The grave lack of a culture of health is well illustrated by diseases such as poliomyelitis, and tuberculosis. In theory, poliomyelitis could be eradicated like smallpox by mass vaccination for all the people in the world or by surveillance containment strategies; in practice, concerted efforts by the WHO had it difficult to find acceptance in some countries, and major resurgences occurred in India and Nigeria, reintroducing polio virus type 1 into more than 20 previously polio-free countries27. By adequate treatment of patients infected by mycobacterium tuberculosis, tuberculosis could be controlled. But, being essentially a disease of poverty, deliberate efforts and continued improvement of social determinants are necessary but were difficult to achieve, particularly in poorer countries27. Although the WHO has made major efforts in this direction and has had much success, without mutual trust and support resulting from a culture of health embracing each and every person these goals probably cannot be attained.\n\n6. Relationship between the two potentials. Interestingly, the proposed type of health leadership and self-management also applies to cultivating the complex relationship between the two potentials (BGP and PAP). An analogous relationship exists in the bond between a horse and its rider. If the rider wants his horse to serve him well, he must take good care of it. The horse needs cleanliness, adequate food and water, an appropriate amount of physical movement, and rest at night in a protected area. In addition, horse and rider must trust each other and the rider must remain in control. Although this analogy with the relationship between a person and his body may be surprising, it illustrates that the two potentials must remain at one with each other. Further features render this relationship important: To a significant part the PAP can compensate for defects of the BGP. This ability is used in rehabilitation. In some conditions it may go quite far. E.g. paraplegic patients may again become independent and professionally active28. When asked, they may even say that they are “healthy”. The compensation of defects in the BGP by the PAP is also important for elderly persons who experience increasing somatic defects. Yet, after having learned to adjust to them, they may experience a sense of well-being and lead a very satisfactory life.\n\nSocial determinants of health are of fundamental importance29. Among other aspects they are complicated by the fact that health care also has an economic component. This has been well known since antiquity. Today, health economy has a much higher and ever increasing impact because health care systems have become more and more costly.\n\n1. Epidemics. The relation between social determinants of health and the PAP becomes vital in case of epidemics, as exemplified by the Ebola crisis in West Africa in 201430. It clearly illustrates the needs for careful investigation of the disease itself, interruption of its transmission, and care of the diseased persons. This means that people have first to understand the disease and what they must do for prevention and for treatment of diseased individuals. They must then become convinced of the purpose of what they do. Last but not least they have to receive the equipment and support they need to become effective. To achieve appropriate handling of diseased individuals, a corresponding organization is essential, particularly because in most cases cultural limitations may have to be overcome. This requires appropriate educational interventions and intense interactions between the responsible organization and the population. An educational programme based on the Meikirch model may be very useful, because it explains the importance of a PAP and gives all participants an identical view about how social determinants of health contribute. Successful cooperation requires agreed upon, common objectives31. The Meikirch model might be most appropriate for this purpose.\n\n2. Tragedy of the commons. In high-income countries many current health care systems offer various services at no or minimal extra costs beyond the ordinary premiums for health insurance. For Germany, the OECD has documented 9.9 average “episodes of care” versus 6.6 for the OECD average of 33 countries, which is the highest in Western Europe. This is not limited, and for each “episode of care” multiple physician contacts are involved32. Patients with compulsory insurance had a mean of 18 physician contacts per year in 2007 (8 physician contacts in the age group 11–15 years; 37 physician contacts in the age group 81–85 years) according to one large insurance company33. Although it is difficult to interpret such statistics, they document overuse in a wealthy country. Hardin has compared such situations to the so-called “tragedy of the commons”34. Whenever a utility is freely available to everybody and paid collectively, it follows logically that everybody tries to get as much out of it as possible. This is no problem as long as the utility is available in excess. The tragedy begins only once the utility becomes the limiting factor. In the past this occurred when farmers used the commons to feed their cows. Every farmer added one or two cows to his allowance. Overgrazing resulted in the tragedy of insufficient feed at the end of the season: the so called “tragic depletion”. Interestingly there is no technical solution to this problem. The tragedy only stopped once the commons were distributed among the farmers as property and each of them had to be satisfied with the amount of land he disposed of. Such a change is, according to theory, often prevented by the “tragic stalemate”: nobody wants to do the first step35. Today such arguments may be applied to the use of health care services, as was observed for the USA36,37. In analogy, the continuous rise in health care costs will not be stopped by appeals and rationing will produce undesired effects. It will become limited only when each patient establishes ownership of his or her health and will assume more responsibility for his or her own health care, including financial consequences. Such a condition might further strengthen health leadership as explained above. Implementation of financial responsibility of individuals for their healthcare, however, will be difficult to introduce. Obviously it must be developed by trial and error without undue hardship for socially disadvantaged groups. Health economists, ethicists and politicians will have to work together. The Meikirch model may facilitate convergence of more or less affluent countries as recommended by the WHO Commission on Social Determinants of Health29.\n\n3. Influence of health care payment models on physician work practice and professional satisfaction of all involved participants. Payment models are designed to produce financial incentives in order to achieve certain goals. Examples are capitation, episode-based and bundled payment, shared savings, pay for performance (PFP), and retainer-based practice38. They are designed from different points of view e.g. to “improve patient care, to preserve or enhance physician professional satisfaction, to satisfy multiple external stakeholders, or to maintain economic viability as business”39. Porter’s proposal e.g. intends to improve “value” in health care40. The proposal is formulated from the point of view of the payer. Other actors in the health care systems may have different points of views41. The most important concern is that payment systems may be manipulated for personal gains. Yet, once patients no longer are sure that their physicians act exclusively in their best interest, the patient-physician relationship is strained42. Today there are many examples of manipulation. Bernard Lown et al.43,44 have shown this phenomenon in cardiology, but other fields of health care are equally vulnerable. The Meikirch model offers a new point of view. In each individual patient the primary aim of a payment system should be to optimally support improvement of health as defined by the model. If all interested parties share this objective, payment systems may be worked out that fully protect the patients interests, serve all partakers, and thereby also are cost-effective45. If these conditions are fulfilled, the payment systems also will lead to professional satisfaction of physicians and other interested parties.\n\n4. Moral hazard. Moral hazard may occur when insurance payments change the behaviours of patients or physicians. This is possible for cases with diseases or treatments that are difficult to assess objectively. Such situations may therefore be exploited by patients in order to get payments46,47. Independent medical evaluations for social security eligibility may then become necessary. The Meikirch model was found to be useful also for such evaluations48. As outlined above, moral hazard undermines mutual trust between diseased persons and physicians. A shared vision of health as set out by the Meikirch model may make it easier for physicians to improve performance and accountability and for patients to truly agree on indications, e.g., for expensive examinations and for surgery40,41.\n\n5. Stressful working conditions. Social determinants contribute to both the demands of life and the two potentials49. For instance, during the industrial revolution the majority of employees had to work very long hours and often in unhealthy settings. Unfortunately, in many low or middle-income countries, such practices have not as yet been stopped. Working conditions evolving from a continuous mutually supporting dialogue between the workers and management may render engagement in a working place more interesting, more flexible, more rewarding and particularly also more productive9. “Economists have recognized that good communication between workers and management, leads to better working conditions and may result in important productivity gains while simultaneously reducing costs50. If such communication includes the Meikirch model, health of the employees might be improved and health care costs reduced.”\n\nThe relationship between social and environmental determinants of health offers another excellent example of the tragedy of the commons. The environment is freely available. Therefore in many places it is used without restrictions and poisoned correspondingly. When the population was small, waste created no problems, and nobody had to pay for it. Population growth, technological developments, and increase in wealth, however, have now led to major problems. In Switzerland, for example, most lakes were polluted to an extent that life in the water became almost extinct. When waste water was cleaned before it was allowed to flow into the lakes, water quality was much improved. Today, a worldwide overproduction of CO2 and other waste products increase greenhouse gases to an extent that the temperatures of the planet will rise. Atomic power plants create an analogous situation. Everybody needs electrical power, yet nobody knows what to do with the atomic waste. In addition, the Fukushima catastrophe has again revealed how dangerous accidents in atomic power plants may be. A further example is exploitation of some fish populations that led to their extinction. Pollution of air, water and soil has serious consequences in many places. Therefore, for the purpose of health, many new agreements and their strict enforcement will be required in order to correct the current untenable situation.\n\nInvestigation of the state of health of an individual by a systems theoretical approach is as yet in its infancy, but scientific methods based on Newtonian physics are inadequate (Table 3 and Table 4)12,13. A new approach should involve individual health as a whole (Figure 1), should consider its history, its energy flow and its sense of purpose.\n\nAs a first approximation the system’s approach comes close to the best of the old fashioned medical history. The patient is to be carefully asked at least the following questions: How was life before onset of the disease? What were the weaknesses and the strengths? How were the conditions around the time of the first symptoms? What was new? What were possible triggering or helping factors at that time and also during further episodes of the disease? How did the patient’s sense of coherence (understandability, manageability and purpose) evolve during the disease?15 What was the purpose in life, and what is it now? How does the patient himself interpret her or his symptoms or condition? How did the patient respond to the different stages of the disease? What could be the most appropriate evolution for the patient and how does she or he see it? These questions may then be modified to try to assess the energy flow, including energies driving preferences of behaviour, values and spirituality. Specific questions about the purpose in life may inform about some of these aspects14,52.\n\nSystems methods may particularly be applied by general practitioners, because very often they have known the patient for years, are also aware of his or her social network and have gone with him or her through previous crises. A long-standing and trusting patient-doctor relationship is a most powerful diagnostic and therapeutic tool53. In many cases modern diagnostic procedures, expensive drugs or seemingly appropriate surgical interventions can be avoided, because they involve risks and encompass unpredictable reactions of the patient’s system. Such restraint may be wise and prevent complications. Reorientation of the patient’s intentions and sense of purpose may induce healing processes. Active participation of the patient is important. Similar considerations apply in rehabilitation institutions, where patients may be observed closely (Table 4).\n\nThis brief allusion reveals that at the present time system’s thinking in medicine is at its very beginning. For appropriate treatment of the PAP much further diagnostic and therapeutic research is needed. Systems theory has a good chance to evolve to a very powerful tool in the coming years.\n\n\nDiscussion\n\nAt the present time the Meikirch model5 is just a hypothesis with new possibilities and new limitations. For these reasons all postulates in this paper await an in depth scientific confirmation. Nevertheless, conclusions derived from the model are supported by observations, logic, and literature giving them already now some validity. Most notably, the Meikirch model fulfils the postulates expressed by the meeting of experts reported by Huber et al.: “ability to adapt and to self-manage”54.\n\nThe concept of health as a complex adaptive system may raise the question as to how medicine has functioned so far without reference to the systemic nature of health. A partial answer may be as follows: Even treatments that are established as being effective do not give the same results in all patients. Different individuals respond in diverse ways to exactly the same procedures. Controlled clinical trials are thus needed to decide about the overall effectiveness of a treatment, but they also show how much individuals may deviate from the mean. To translate study results into decisions for individual patients it has always been an established practice of physicians to carefully consider medical histories and to follow up each patient in order to recognize possible developments in an undesirable direction. Systems theory gives these empirical practices a theoretical basis.\n\nWhen looking at the Meikirch model it appears that the PAP offers great opportunities to do much more for health than has been done so far. Since the publications of Virchow in 1858 health and disease were considered to be properties of the physical body55. There were some good reasons for this assumption: Ordinary infectious diseases, many cancers and the sequelae of accidents have clear physical, chemical, or microbiological causes and manifestations. Whenever these causes could be eliminated, corresponding improvements in health were the result. On the other hand physicians are aware that many patients suffer from important symptoms that cannot be explained adequately by objectively recognizable signs of disease. Others have distress that is distinctly more severe than in most patients who suffer with the same physical condition. There are also patients who do not complain even though they are severely diseased. These observations support the concept that the state of health of each patient should truly be regarded as a complex adaptive system. This feature allows a new look at symptoms and offers new possibilities for treatment. For example, solutions found in past crises may teach the patient and the physician new therapeutic options for current problems. In some cases diseases may serve as a learning opportunity on how to conduct one’s life. We also think that psychological support for the creation of a new future for a patient may have a high therapeutic value. A positive belief of the physician in the patient's abilities may be very valuable to readjust himself with his whole system toward a better state of health.\n\nAn interesting new aspect of the Meikirch model is that it may serve as a joint vision for all people who are personally or professionally concerned with health, be it patients, physicians, nurses, administrators, health workers in any profession, journalists, business people or politicians. A common goal is particularly important for departmental, interdepartmental and intersectoral cooperation. In the future, a shared vision for all people based on the Meikirch model may be a great asset that strengthens public health and medicine31. The model may also serve well to interpret article 25 of the Universal Declaration of Human Rights (1948)56 and article 12 of the International Covenant on Economic, Social and Cultural Rights (1976)57. Use of the five components and ten complex interactions may help to better understand the social structure of some health problems. A good example is the increasing prevalence of patients with burnout. This condition is initiated by current types of management procedures designed to stimulate employees to the extent that they give their maximum or even more. Consequently, their demands of life are pushed up as much as possible and a rising number break down. Thereafter health and social systems have to take care of them. This implies that one part of the society, i.e., business, creates a problem and another part of the same society, i.e., social security, has to pay for it. With the help of the Meikirch model such unethical situations can be recognized easily and may lead to corrections that obviously must occur at a political level.\n\nOur observation is that today’s health care systems have in part been distracted from their main mission by rising health care costs. This corresponds surprisingly well to the tragedy of the commons34. It applies equally to patients and physicians and leads to excessive amounts of investigations and operations44. Furthermore, moral hazard may aggravate the difficulties46,47. All parties involved should be reminded that initially, i.e., in the past, they made a contract for health and not for a maximum of services or of income. How to balance health care with limited financial resources is an interdisciplinary dilemma that cannot be solved by individual health workers, administrators, or patients. In each context policies that successfully combine scientific, economic, and normative postulates must be worked out among all stake holders. In the past, the lack of a common concept of health limited the success of such interdisciplinary and intersectoral groups. We trust that the Meikirch model will improve procedures to solve these problems.\n\nSome confusion has occurred in the past about the importance of the relative contributions to people’s health by public health activities or by individual medical care. Unfortunately, mutual awareness and respect between the two approaches to health have remained limited. Interestingly, such situations may be understood better by “Russell’s paradox”58. Bertrand Russell found that the combination of observations at two different levels may lead to incompatibilities. A good example for an intuitive understanding is the following statement: “The world is bad! Everybody cares exclusively for himself! Only I care for myself!” In the health care system Russell’s paradox becomes apparent when looking at parameters such as health and disease: When investigated by the Meikirch model an individual can be healthy or diseased. In contrast, most groups of people or populations can neither be healthy nor diseased. Only a portion of the investigated individuals is either healthy or diseased. Therefore public health works with indicators of health or disease such as longevity, live birth rate, mortality rate, etc. and expresses them in statistical numbers. Interestingly, a public health intervention such as vaccination in a group of people is likely to reduce the prevalence of the disease. Yet some individuals may contract the disease and will not experience the positive effect of their vaccination. Consequently vaccination is effective when analysed in groups, but not necessarily in individuals. The patient-physician interaction is important in individual medicine, but not in public health. We conclude that mixing judgments of individual medical care with judgments of public health - including comparisons of their relative contributions to the health of a population - is not purposeful and must be avoided. Indeed, they simply complement each other. Some reluctance to support the Meikirch model in the health literature may presumably be explained by Russell’s paradox59–61.\n\nThe Meikirch model implies that for each individual demands of life and two potentials are critical for health. The high interest in natural sciences has led to an impressive machinery for the improvement of the BGP. In contrast, the PAP with its non-technical aspects has by far not received the same attention. We conclude that current medicine and public health will be able to offer much more health support, once the importance of the PAP is generally recognized, further developed, and fully integrated into all health care activities. The Meikirch model offers completely new opportunities; this applies to diverse settings at local and global levels and in low and high income countries. This new view of health and wellbeing may also be interesting for the interpretation of the Universal Declaration of Human Rights56, the International Covenant on Economic, Social and Cultural Rights57 and the General Comment No. 14 related to art. 12 of the International Covenant62. In principle the Meikirch model is applicable in any situation where a concern for health is relevant.", "appendix": "Author contributions\n\n\n\nBoth authors contributed to drafting the work, 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 Dr Wolfram Goessling for reviewing the manuscript and to Dr Arthur Funkhouser for improving the English language of the paper.\n\n\nReferences\n\nSwiss Federal Office: Swiss Federal Office of Statistics, personal communication. Neuchatel, Switzerland, 2014.\n\nWHO: Global Action Plan for the Prevention and Control of Noncommunicable Diseases 2013–2020. 2013. 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PubMed Abstract \| Publisher Full Text\n\nFrenk J, Gómez-Dantés O: Designing a framework for the concept of health. J Public Health Policy. 2014; 35(3): 401–6. PubMed Abstract \| Publisher Full Text\n\nOzonoff D: Choosing health. J Public Health Policy. 2014; 35(3): 407–10. PubMed Abstract \| Publisher Full Text\n\nCommittee on Economic, Social and Cultural Rights – Report on the Twenty Second, Twenty Third and Twenty Fourth Sessions. IV. General Comment No. 14 (2000): The right to the highest attainable standard of health (art. 12 of the International Covenant on Economic, Social and Cultural Rights). (09.02.2016). Page 128. Reference Source\n\nBlack DS, Cole SW, Irwin MR, et al.: Yogic meditation reverses NF-κB and IRF-related transcriptome dynamics in leukocytes of family dementia caregivers in a randomized controlled trial. Psychoneuroendocrinology. 2013; 38(3): 348–55. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGulliksson M, Burell G, Vessby B, et al.: Randomized Controlled Trial of Cognitive Behavioral Therapy vs Standard Treatment to Prevent Recurrent Cardiovascular Events in Patients With Coronary Heart Disease: Secondary Prevention in Uppsala Primary Health Care project (SUPRIM). Arch Intern Med. 2011; 171(2): 134–40. PubMed Abstract \| Publisher Full Text\n\nSouza JG, Apolinario D, Magaldi RM, et al.: Functional health literacy and glycaemic control in older adults with type 2 diabetes: a cross-sectional study. BMJ Open. 2014; 4(2): e004180. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGillett M, Royle P, Snaith A, et al.: Non-pharmacological interventions to reduce the risk of diabetes in people with impaired glucose regulation: a systematic review and economic evaluation. Health Technol Assess. 2012; 16(33): 1–236, iii–iv. PubMed Abstract \| Publisher Full Text\n\nWolf MS, Gazmararian JA, Baker DW: Health literacy and functional health status among older adults. Arch Intern Med. 2005; 165(17): 1946–52. PubMed Abstract \| Publisher Full Text\n\nRuss TC, Stamatakis E, Hamer M, et al.: Association between psychological distress and mortality: individual participant pooled analysis of 10 prospective cohort studies. BMJ. 2012; 345: e4933. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOlivares-Tirado P, Leyton G, Salazar E: Personality factors and self-perceived health in Chilean elderly population. Health. 2013; 5(12A): 86–96. Publisher Full Text\n\nChochinov HM, Hack T, Hassard T, et al.: Dignity therapy: a novel psychotherapeutic intervention for patients near the end of life. J Clin Oncol. 2005; 23(24): 5520–5. PubMed Abstract \| Publisher Full Text\n\nParikh RB, Kirch RA, Smith TJ, et al.: Early specialty palliative care--translating data in oncology into practice. N Engl J Med. 2013; 369(24): 2347–51. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "12994", "date": "31 Mar 2016", "name": "Amitav Banerjee", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis opinion paper titled, \"Understanding the nature of health: New perspective for medicine and public health. Improved wellbeing at lower costs\" uses number of illustrative examples on how the Meikirch Model proposed by the lead author in a previous publications can be used as a strategic tool to cope with health and disease burdens in all societies. The concepts enunciated in the Meikirch Model has some common points in the traditional levels of prevention and epidemiological triad model put forward by Leavell and Clark in the 1940s1. For instance, the biological potential can be compared to the host factors in the epidemiological triad, agent and environment are self explanatory in the epidemiological triad. Environment also is an important component of the Meikirch Model. While early diagnosis and treatment may be more in line with curative medicine, the level of Tertiary Prevention as proposed by Leavell and Clark has a lot in common with Personally Acquired Potential (PAP) in the Meikirch Model. Psychological Rehabilitation, Social Rehabilitation, Vocational Rehabilitation, Medical Rehabilitation are the activities advocated in Tertiary Prevention which have a lot in common with PAP. Perhaps the authors, may like to bring out these similarities, and in what way the Meikirch Model goes a step further to push the agenda towards empowerment of individuals and communities towards their health promotion by active participation and assuming individual & Community responsibility. This may facilitate a seamless transition in paradigm from the earlier model proposed by Leavell and Clark and the Meikirch Model. Authors may emphasize how the Meikirch Model has a more positive approach compared to the previous model of health promotion and prevention. In times of rising medical costs the the Meikirch Model can be an important tool and point of reference for policy makers and other stakeholders.", "responses": [] }, { "id": "12424", "date": "18 Apr 2016", "name": "Joachim P Sturmberg", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nUnderstanding the nature of health: New perspectives for medicine and public health. Improved wellbeing at lower costs is an attempt to highlight the interconnected nature of health between the individual and his “lived environment”. It is an important step towards reframing 1 the necessary societal discourse about the nature of health and its implications for personal and professional care and the organisation of healthcare in one’s society. The authors are commended for making these complex tasks understandable and manageable for patients, practitioners and policy makers. In particular they make compelling arguments why both users and providers of health care need to come together and “renegotiate” the purpose of healthcare and define mutual expectations from the health system.\n\nFollowing are a number of thoughts stimulated by reading the article for consideration in future revisions of this paper. (1) Understanding the nature of health. There has been important other work done in this domain, e.g. Macklem 2, Jylhä 3, Jadad 4, Lewis 5, Sturmberg 6,7 and Sturmberg et al 8. Importantly the “experiential nature of health”, pointing to the importance of adding a cognitive domain to the definition of health, deserves more explicit mention. A review of the “health definition” literature highlights that the experiential aspect is persistent and central across time, philosophical traditions and cultures 9-21. The authors only refer to its importance in indirect ways: The final psychosocial demand [which in reality is a cognitive ability] is to achieve peace with the fact that every human must die.A well-founded sense of purpose in life strengthens it [personally acquired potential] also.A high PAP [personal adaptive potential] is important for health. In some cases diseases may serve as a learning opportunity on how to conduct one’s life.[health as an] ability to adapt and to self-manage. The importance of the cognitive domain for health is also reflected in the development of subcultures in health, usually at the extreme of the spectrum like drug use as self-therapy for mental health experiences or the obsessive consciousness on health in every action of daily living. Understanding health as “experiential and in one’s one context” is further reflected in health inequality. It is “relative” inequality that matters most, as stated in the paper: The social determinants of health have a very important enabling or inhibiting function. Wilkinson and Pickett identified better health of people in countries with less inequality of incomes. Longevity is not solely related to people’s income, but is also strongly affected by their autonomy and social participation, which are major determinants of health. (2) Economics. While there is no doubt that economics play an important role in financing health, the statement Today, health economy has a much higher and ever increasing impact because health care systems have become more and more costly. reflects a one-sided a view. It echoes the economic rationalist [linear] paradigm. In the context of this paper, it needs to be debunked. Principally a ledger has two sides – income and expenditure; more importantly though, what is an expense in one ledger also is an income in another ledger. Currently politicians, bureaucrats and financiers put forward rather one-eyed arguments to justify “cut and burn” approaches in the reform efforts of our healthcare systems. The “unintended” but highly foreseeable consequences are degradations of social and environmental conditions so necessary to the “production of good health” 22. (3) Greater emphasis? Epigenetics. In the context of “dynamics of health” it appears essential to include the growing evidence of the important aspects of epigenetics and in particular those of social epigenetics 23. These insights are important in understanding variation in disease expression in various communities. Equally, they explain disease distributions in communities but more importantly that health improvement strategies may take a few generations to show reversals in disease distributions. Genetic and physiological network interactions. There is a need to at least hint to the “low level mechanisms” that explain the observations between the environmental, social, emotional and cognitive perturbations on health 24,25. These insights satisfy “the scientific need of argument” for various players to engage in “system’s based” decision-making and strengthen the concluding remarks: In each context policies that successfully combine scientific, economic, and normative postulates must be worked out among all stake holders. The limitation of controlled trials. The basic assumption underpinning controlled trials are inconsistent with “complex adaptive systems” frameworks. Complexity entails nonlinearity and variability, two features of nature that cannot be controlled for 26 making inferences from controlled trials tenuous. Tools like neural network or cluster analysis provide the insights that are really needed in personal care and population health decision-making. (4) Changing the worldview. I think it is fair to state that “modern medicine” has lost its soul – medicine was always primarily about care rather than cure – just consider Archie Cochrane: Cure is rare but the need for care is widespread 27. The power of care, linking back to above mentioned “physiological networks”, is greatly underestimated and its importance is largely missing from the medical curriculum. The authors’ comments in this regard: We also think that psychological support for the creation of a new future for a patient may have a high therapeutic value. A positive belief of the physician in the patient’s abilities may be very valuable to readjust himself with his whole system toward a better state of health. cannot be overemphasised. Care leads to self-care which ultimately leads to better “experiential health”, independent of the presence or absence of identifiable pathologies. It is right to emphasise that a systems medicine approach is particularly attractive to general practitioners – who I believe have always tacitly embraced the notion and practiced accordingly – it must be actively promoted by all involved in providing care to individuals and those having the responsibilities to planning and managing health systems.", "responses": [] } ]	1	https://f1000research.com/articles/5-167
https://f1000research.com/articles/5-166/v1	12 Feb 16	{ "type": "Research Article", "title": "Improving quality for maternal care - a case study from Kerala, India", "authors": [ "Ioana Vlad", "VP Paily", "Rajeev Sadanandan", "Françoise Cluzeau", "M Beena", "Rajasekharan Nair", "Emma Newbatt", "Sujit Ghosh", "K Sandeep", "Kalipso Chalkidou", "VP Paily", "Rajeev Sadanandan", "Françoise Cluzeau", "M Beena", "Rajasekharan Nair", "Emma Newbatt", "Sujit Ghosh", "K Sandeep", "Kalipso Chalkidou" ], "abstract": "Background: The implementation of maternal health guidelines remains unsatisfactory, even for simple, well established interventions. In settings where most births occur in health facilities, as is the case in Kerala, India, preventing maternal mortality is linked to quality of care improvements.\nContext: Evidence-informed quality standards (QS), including quality statements and measurable structure and process indicators, are one innovative way of tackling the guideline implementation gap. Having adopted a zero tolerance policy to maternal deaths, the Government of Kerala worked in partnership with the Kerala Federation of Obstetricians & Gynaecologists (KFOG) and NICE International to select the clinical topic, develop and initiate implementation of the first clinical QS for reducing maternal mortality in the state.\nDescription of practice: The NICE QS development framework was adapted to the Kerala context, with local ownership being a key principle. Locally generated evidence identified post-partum haemorrhage as the leading cause of maternal death, and as the key priority for the QS. A multidisciplinary group (including policy-makers, gynaecologists and obstetricians, nurses and administrators) was established. Multi-stakeholder workshops convened by the group ensured that the statements, derived from global and local guidelines, and their corresponding indicators were relevant and acceptable to clinicians and policy-makers in Kerala. Furthermore, it helped identify practical methods for implementing the standards and monitoring outcomes.\nLessons learned: An independent evaluation of the project highlighted the equal importance of a strong evidence-base and an inclusive development process. There is no one-size-fits-all process for QS development; a principle-based approach might be a better guide for countries to adapt global evidence to their local context.", "keywords": [ "maternal health", "quality improvement", "evidence-informed policy-making" ], "content": "The challenge\n\nFor low and middle-income countries (LMIC) moving towards Universal Health Coverage, social protection and the expansion of access to essential health services need to be balanced with efforts to ensure quality of care1. In the case of maternal health, addressing the “quality care gap” for births occurring in health facilities is central to achieve the promised reductions in maternal deaths by implementing the available effective and cost-effective interventions2. However, implementation of global clinical guidelines remains unsatisfactory, even in the case of basic, well established interventions3,4. In this context, there have been calls for strengthening the evidence base on the implementation of such interventions in LMIC settings, including in terms of feasibility and replicability5.\n\nThis paper describes the partnership between NICE International, a division of the National Institute for Health and Care Excellence (NICE), United Kingdom, the Government of Kerala, India, and the Kerala Federation of Obstetrics and Gynaecology (KFOG) in developing and implementing the first clinical quality standards (QS) for reducing maternal mortality in the state, based on robust clinical guidelines. It thus explores the complex process of developing and implementing evidence-based QS in a LMIC setting and, more significantly, the difficulty of embedding QS in quality improvement processes within the health system.\n\nQuality standards are a concise sets of prioritised statements designed to drive measurable quality improvements6. They provide “health policymakers, health insurers, service providers, healthcare professionals and patients with definitions of what high quality healthcare looks like in practice; and related performance measures that are reliable and meaningful to the local setting in which they are used”7. As such, the development and implementation of QS are highly context-specific. By presenting the process of developing and implementing QS in a LMIC context, this paper highlights lessons learned for similar settings, while acknowledging that the process cannot be simply transferred from setting to setting. In doing so, the paper also reflects on global health partnerships and on the sustainability of evidence-informed decision-making processes established through such partnerships8. Box 1 summarises the key messages of this paper.\n\n1. In the development of QS in LMIC contexts, local buy-in for the process of development as well as for the end product bear equal importance.\n\n2. There is no one-size-fits-all process for QS development - instead, a principle-based approach might be a better guide for countries to adapt global evidence to their local context.\n\n3. Agencies like NICE International, which provide technical support for evidence-informed decision-making processes, will maximise their effectiveness if the local demand and the capacity for such processes (and their products) is stimulated and policy makers fully engaged.\n\n\nBackground to the partnership\n\nNICE International started engaging with the Indian state of Kerala in 2009, at the invitation of the then Minister and Principal Secretary for Health, with a workshop organised by the state’s Ministry of Health. The workshop was designed as a multidisciplinary consultation around priority areas for the Kerala Ministry of Health, related to quality of care: quality standards and standard operating procedures for hospital providers; standard treatment guidelines (STGs); and approaches to implementation of STGs and clinical pathways. What followed was an exploratory period including several visits to NICE’s headquarters by Kerala policy-makers and characterised by the creation of informal networks with individuals in leadership positions from government bodies and professional organisations in Kerala. This period of informal engagement culminated in a project conceived to support the Government of Kerala in the development and implementation of QS for preventing maternal mortality, a policy priority for the state.\n\nAlthough Kerala has the lowest maternal mortality rate (MMR) in India and has achieved the associated Millennium Development Goal targets9, its government had pledged to reduce it further, aiming at a 50% decrease in MMR by 2017, the end of its 12th five year plan on medical and public health10. Table 1 outlines key indicators related to maternal health in Kerala, by comparison to the national level.\n\n*2010–12 Sample Registration Survey §The World Bank, 2013.\n\nWHO, UNICEF, UNFPA, The World Bank, & UN Population Division Maternal Mortality Estimation Inter-Agency Group; modelled estimate, 2014.\n\nThe priority given to maternal health by the Government can be explained by the fact that, compared to other health indicators, such as life expectancy and infant mortality rate, the MMR had remained relatively high, with no significant improvements in recent years. Ninety eight per cent of deliveries in Kerala take place in institutions, suggesting that efforts to reduce avoidable maternal deaths should focus on improving care at health facilities11. Out of the over 500 000 deliveries registered in Kerala annually, approximately 72% take place in private facilities, and 28% in public government teaching and non-teaching hospitals12. The high rates of institutional delivery, low fertility and high female literacy suggested that reductions in avoidable maternal deaths are possible through improvements in quality of care.\n\n\nMethods\n\nThe formal partnership between the Government of Kerala, represented by the Principal Secretary for Health and Family Welfare, the KFOG and NICE International was launched in 2012. It resulted in ten QS related to management and prevention of post-partum haemorrhage (PPH) (QS 1-5) and hypertension in pregnancy (QS 6-10). The QS were launched in January 2013, in the presence of the UK Health Minister and the Principal for Health in Kerala.\n\nLocal ownership was a core principle of the QS development process, with the Government of Kerala and KFOG taking a leadership role. NICE International’s contribution was to (a) provide a technical and methodological framework for the development of the QS; (b) support the institutional partnerships between the Government of Kerala, the KFOG and other local stakeholders.\n\nIn terms of technical and methodological support, the NICE framework and processes that underpin development of QS for the UK National Health Service (NHS) were used. This includes the selection of high-priority topics for quality improvement in a defined service area, and combines evidence-based guidance with stakeholder priorities and evidence of current practices through a deliberative process (ran by a QS advisory committee), ultimately resulting in a series of quality statements and corresponding measurable indicators13. For the NHS, as for the Kerala health system, developing QS is a highly contextualised process. As such, the NICE framework was adapted to fit the local context, by identifying institutional decision-making rules, key stakeholders and health system functioning characteristics in Kerala.\n\nLocally generated evidence played a key role, particularly in the identification of the leading causes of maternal death in Kerala and therefore the key priorities for the QS. The process was fundamentally informed by the Confidential Review of Maternal Death (CRMD) Audit, which has been run by the KFOG since 2004 and is the only one of its kind in India11,12. As shown in Figure 1, PPH and hypertension are the two main causes of maternal mortality. Furthermore, they are estimated to consistently account for between 29–44% of maternal deaths in Kerala between 2006–200914.\n\nAdapted from the 2nd edition of the CRMD12.\n\nTogether with the findings of the CRMD, local epidemiological data and routinely reported systems data (e.g., the Sample Registration System conducted by the Office of the Register General) were assessed using deliberative multi-stakeholder processes convened with mentorship from NICE International. This mentorship consisted of a total of eight workshops (four during the development period and four during implementation). The multi-stakeholder workshops that comprised the QS development process functioned to support the creation and maintenance of institutional links, most notably between KFOG and the state’s Government. Following an initial workshop in June 2012, focused on the active management of the third stage of labour (one of the ten statements included in the first edition of the finalised standards), a multidisciplinary group was established to lead on the development of the standards, the wider consultation process for each statement, and the implementation of the QS. Members included key policy-makers (the Principal Secretary, the Director of the State's National Health Mission-NHM) and leading gynaecologists and obstetricians, nurses and administrators from across Kerala.\n\nThe group developed quality statements derived from evidence-based guidelines published by the KFOG, NICE, the World Health Organization, and the UK Royal College of Obstetricians and Gynaecologists (see Box 2 for an example of a quality statement and its link to global guidelines), but adapted based on the experience of practising obstetricians and nurses in Kerala.\n\nThe QS implementation required action ranging from monitoring (antenatal and labour), to administering drugs and blood products, to referral procedures. These actions raised implementation needs related to data collection mechanisms, on the one hand, and state-wide implementation support mechanisms, on the other. Through the consultation processes, the multi-stakeholder group identified practical methods for implementing the standards and monitoring outcomes. The implementation plan, including an initial needs assessment, identified the need for staff training, as well as for upfront investment in infrastructure, improvements of drug availability and the need to support clinical audit. All 400 staff working in the maternity wards of the pilot health facilities participated in an initial training carried out by the KFOG. The Government of Kerala allocated the funds needed to cover these needs for the pilot period, as per the core tasks outlined in Box 3.\n\nImplementation pilots started in six public and two private maternity hospitals across Kerala in April 2013. In line with the plan, pilot hospitals collected monthly data on the QS indictors from their delivery registers and sent them to the NHM for analysis. These data were discussed in monthly review meetings chaired by the NHM Director, where the staff from the pilot maternities, members from the KFOG and technical staff from the NHM reviewed progress reports and provided feedback on the process of implementing the standards. These meetings were designed to create a feedback loop supporting implementation.\n\n\nResults\n\nAn independent, qualitative evaluation of NICE International’s engagement in Kerala was conducted in order to shed light on the lessons learned following a year of QS implementation (2013–2014). Drawing on key informant interviews and a document review, the assessment focused on participants’ perception on the overall value of the QS, as well as how the staff training helped implement the QS and improve practice. The evaluation also examined the extent to which similar initiatives had been taken up in other locations and whether new, similar partnerships were developed by NICE International in India. The focus on perceptions about the process highlights the importance placed by NICE International on the local ownership of the project15.\n\nWith regards to the overall value of the QS, participants valued the deliberative development process as being critical in producing statements and indicators that were relevant and acceptable to both clinicians and policy-makers in Kerala. This deliberative approach, which participants indicated was innovative for producing QS in Kerala, was seen as having maximised the likelihood of implementation, especially given that the major partners in developing the QS were also involved in piloting. There was particular value placed on the fact that NICE International’s approach is non-prescriptive, thus allowing for the QS development and implementation to be locally owned and driven by Kerala institutions. It thus resulted in guidance that was perceived as detailed and explicit, compared to other more general sources of guidance. However, despite a high buy-in for the implementation process, there remained a perceived tension between providing standardised care and professional freedom. Consequently, it remains to be seen whether similar level of acceptance would be replicated when the QS are scaled up across Kerala, when it is expected that the main value of the QS would stem from their perceived role in improving quality of care and/or clinical outcome.\n\nWith regards to implementation, the QS were perceived as a valuable tool to improve and standardise quality of care, exemplified by changes in practice such as: the introduction of management of fourth stage of labour; the use of sterile delivery kits; greater consistency in the management of the third stage of labour and the use of oxytocin; the measurement of blood loss instead of subjective estimation; and better record keeping. Furthermore, staff from the sampled pilot maternities reported high satisfaction with the QS and in some cases increased confidence in PPH management when following the standards.\n\nChallenges referred to insufficient staffing in some of the pilot hospitals, on the one hand, and variability in practice, on the other. Specifically, the independent evaluation suggested a lack of sufficient staff for managing fourth stage of labour and variations in some of the actions indicated by the QS, i.e., use of magnesium sulphate to prevent eclampsia and of urine protein testing. This highlights the importance of standard operating procedures for each facility, which need to be updated during and beyond the piloting phase. These standard operation procedures refer to the need that each facility consider the QS implementation in term of staff training needs, drug supply, referral systems and staffing for the needed monitoring14. Furthermore, the importance of staff training was highlighted as key for the sustainability of the implementation, particularly given the observed high turnover of labour room staff. Pilot data suggested that a refresher of the initial staff training provided by KFOG is needed in order to respond to the requirements of implementation and ensure its sustainability. Plans for ongoing training, under discussion at several review meetings, highlighted the need to target both central level staff (to ensure continued support for the implementation), as well as clinicians and nurses (to account for staff turnover and as a refresher).\n\nAnother important lesson learned stems from the lack of baseline data (which was discussed at the workshops, but not collected during implementation), as it would have allowed a quantitative assessment of quality improvements. A related issue was that the compilation of data in the maternity wards and the feedback loop between the NHM and the pilot maternities has not functioned as well as was expected. For example, the data recorded in the labour registers designed for implementing the QS were not analysed systematically in order to identify needed adjustment to the implementation. In addition, few facilities carried out clinical audit, which would have provided an opportunity for staff to reflect on their practice. As a result, the monthly meetings between the NHM and facilities identified deficiencies in monitoring improvement in the process of care. This highlights the need to strengthen local data analysis capacity, as well as ongoing staff training throughout the implementation of the QS, whether at pilot level or for scale up.\n\nThe Kerala experience up to this point illustrates a process of engagement with top-level policy-makers that built on global and existing local evidence and led to the implementation of QS at facility level. In recording elements of this experience, this paper provides an account of the process of complex decision-making informed by evidence in a “real life” situation in a LMIC setting, across different institutional levels of the health system, with international support, but limited international funding for implementation.\n\nA major takeaway from this process, which is ongoing, refers to the importance of engaged professional and policy leaders who are able to promote rapid change in the community, with a high level of visibility and acceptability. However, there is a danger that the continuity of the process may be affected when key individuals change posts and responsibilities and their initial input cannot be sustained. NICE International’s engagement in Kerala aimed to support sufficiently robust institutional links for the QS implementation process to continue when unavoidable changes in leadership happened. As a complex health system intervention, future will tell the extent to which this has been successful. It is encouraging that the Government of Kerala decided to expand the implementation of the QS to another 32 health facilities in Kerala. Furthermore, the process for developing the standards has garnered interest at state level and across India. The Government of Kerala completed QS for neonatal care in 2015, recently pledging Rs. 25 crore (USD4 million) for their implementation in 22 hospitals as well as expressing interest in their applications to antenatal care. Combined staff training is now taking place to implement both the maternal and neonatal QS in these facilities.\n\nSimilarly, there are nascent plans to draw on the QS development processes in other Indian states and at central level. Government officials in other states (Bihar, Odisha), having visited Kerala and interacted with NICE International, have expressed interest in the CRMD and QS process. Furthermore, the Rashtriya Swasthya Bima Yojna (RSBY), the government health insurance scheme for population under the poverty line, commissioned the development of clinical pathways for seven medical conditions, using principles for evidence-informed decision-making that were at the core of the QS development process, with the goal to eliminate the practice of wasteful and expensive medical procedures. Further, the Ministry of Health and Family Welfare (MoHFW) has commissioned the development of twelve Standard Treatment Guidelines that include Quality Standards for twelve health conditions, using a similar framework to the one used in Kerala.\n\nHowever, the independent assessment carried out by Itad15 suggests that some stakeholders valued the QS more as a product, rather than as a process that can be scaled up and replicated. This difference in perceptions regarding replicability highlights the importance of local buy-in for the principles of evidence-informed decision-making, and not only for its products (in this case, the QS). The process of participative development needs to be carried out in each specific context. Even if the product turns out to be similar across contexts, local input in the process is too vital to be bypassed, especially with regards to the feasibility of implementation. Recognizing this, NICE International has developed a guide containing principles (and not a standardised process) for developing QS in LMICs, using examples based on the experience in Kerala and suggesting ways of adapting such principles to diverse local contexts7.\n\nGlobal health partnerships as the one presented here, need to support institutionalised processes of evidence-informed decision-making at local level; this is critical for sustainability, especially when faced with unavoidable changes in leadership. While informal networks can initially help establish such partnerships, this desirable shift from informal to institutional links promotes good governance in global health partnerships16. Organisations like NICE International, which provide technical support for evidence-informed decision-making processes, will maximise their impact if the local demand and the capacity for such processes (and their products) is stimulated and constantly engaged.", "appendix": "Author contributions\n\n\n\nVPP, RS, FC, MB, RN, KS, KC were responsible with the conception and design of the work, as well as with data acquisition and analysis. IV, EN, SG contributed to the acquisition, analysis and interpretation of the data. IV and FC wrote the first draft of the article. All authors revised the article for intellectual content and gave final approval of the version to be published.\n\n\nCompeting interests\n\n\n\nMs Newbatt is an employee of Itad, who were contracted by NICE International on a consultancy basis to undertake an assessment of their work in India (cited within the submitted manuscript). However, Itad was contracted as an independent evaluator and therefore were not subject to any undue influence.\n\nDr Ghosh was also involved in the independent evaluation. There were no competing interests or undue influence.\n\nDr Sadanandan was the Secretary in charge of Health in Kerala when the intervention that is the topic of the paper was implemented. Dr Sadanandan held the original discussion with the collaborating organisation, arranged resources, granted the needed permissions and had oversight over its implementation.\n\nAll other authors have declared that no competing interests exist.\n\n\nGrant information\n\nThe work was funded by a grant to NICE from the Health Partnership Scheme of the Department for International Development (DFID) (reference: Pilot 1).\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nWorld Health Organization: Research for universal health coverage: World Health Report 2013. Geneva: World Health Organization; 2013. Reference Source\n\nTamburlini G, Siupsinskas G, Bacci A: Quality of maternal and neonatal care in Albania, Turkmenistan and Kazakhstan: a systematic, standard-based, participatory assessment. PLoS One. 2011; 6(12): e28763. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHeiby JR, Armbruster D, Jacobs TA: Better care for every patient, every time: improving quality in low health systems. BJOG. 2014; 121(Suppl 4): 4–7. PubMed Abstract \| Publisher Full Text\n\nNtoburi S, Hutchings A, Sanderson C, et al.: Development of paediatric quality of inpatient care indicators for low-income countries - A Delphi study. BMC Pediatr. 2010; 10(1): 90. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBhutta ZA, Salam RA, Lassi ZS, et al.: Approaches to improve quality of care (QoC) for women and newborns: conclusions, evidence gaps and research priorities. Reprod Health. 2014; 11(Suppl 2): S5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNational Institute for Health and Care Excellence (NICE): Standards and Indicators. Quality Standards, London: NICE; 2015. Reference Source\n\nNICE International: Principles for developing clinical Quality Standards in low and middle income countries. London: NICE International; 2014. Reference Source\n\nChalkidou K, Levine R, Dillon A: Helping poorer countries make locally informed health decisions. BMJ. 2010; 341: c3651. PubMed Abstract \| Publisher Full Text\n\nChatterjee A, Paily VP: Achieving Millennium Development Goals 4 and 5 in India. BJOG. 2011; 118(Suppl 2): 47–59. PubMed Abstract \| Publisher Full Text\n\nGovernment of Kerala: Twelfth Five Year Plan (2012–2017). Working Group Report on Medical and Public Health, Thiruvananthapuram: State Planning Board; 2011. Reference Source\n\nPaily VP, Ambujam K, Rajasekharan Nair V, et al.: Confidential Review of Maternal Deaths in Kerala: a country case study. BJOG. 2014; 121(Suppl 4): 61–6. PubMed Abstract \| Publisher Full Text\n\nKerala Federation of Obstetrics and Gynaecology: Second Report of Confidential review of Maternal Deaths, Kerala. Why mothers die, Kerala 2006–2009. Kerala, India. Reference Source\n\nNational Institute for Health and Care Excellence (NICE): Quality standards process guide. London: NICE; 2014. Reference Source\n\nWilkinson T: Improving Maternity Care in Kerala: Quality Standards on Post-partum Haemorrage and Hypertensive Disorders of Pregnancy. Estimate budget impact and lives saved. London: NICE, 2015; Unpublished report.\n\nItad: Report on NICE International’s Engagement in India. Hove: Itad; 2015. Reference Source\n\nParkhurst JO: Support local governance to get research into policy. In: SciDevNet. 2011. Reference Source" }	[ { "id": "12582", "date": "22 Feb 2016", "name": "Nerges Mistry", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 study by Vlad et al. is a significant example how quality initiatives are nested within the public health system of Kerala – a “health iconic” State of South India. The representativeness of this case study for other States in India (particularly those with weak health systems and poor socio-economic indices) is possibly quite weak and needs to have a cautionary mention in the discussion. The uniqueness of the components of the Kerala initiative need to be highlighted. In my opinion these would comprise of (a) the significant and dedicated contribution of private participants and the KFOG in persistently facilitating the quality initiatives yet placing it in the hands of the public health sector (b) the wise buy in of the Government of Kerala of international evidence based initiatives and for forging a symbiotic relationship with the private sector (e) facilitation of completion of approach through implementation processes such as definition of quality benchmarks and facility level training. It is hoped that the pioneering efforts of Kerala find wide resonance in other parts of India", "responses": [] }, { "id": "14606", "date": "27 Jun 2016", "name": "Marleen Temmerman", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nInteresting paper addressing an important public health topic. Well written and contains methods and models that could be replicated and implemented in different settings.", "responses": [] }, { "id": "14519", "date": "06 Jul 2016", "name": "Rachel Foskett-Tharby", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 of a quality improvement initiative which highlights the importance of such activities being piloted and refined prior to widespread implementation. I have a couple of minor comments:\nFirstly, given that the evaluation appeared to cover the full quality standard it would be useful for readers to be able to review the full list of QS statements. Could this be added as a table or supplementary information?\n\nSecondly, the first paragraph of the results describes the methods of the evaluation. The authors may wish to move this to methods section and provide more detail.\n\nThirdly, it would be useful to have some more information about the participants in the evaluation e.g. roles, participation in the development of the QS as this would provide some context for the findings.", "responses": [] } ]	1	https://f1000research.com/articles/5-166
https://f1000research.com/articles/4-1442/v1	14 Dec 15	{ "type": "Research Article", "title": "The representation of research in the national curriculum and secondary school pupils’ perceptions of research, its function, usefulness and value to their lives", "authors": [ "Kay Yeoman", "Laura Bowater", "Elena Nardi", "Laura Bowater", "Elena Nardi" ], "abstract": "Young people’s views on research, how it’s conducted and whether it’s important, influences the decisions they make about their further studies and career choices. We investigate how research is represented within the English national curriculum and the examination boards because we recognise that what is being taught influences secondary pupil views on research. We use questionnaire data to focus particularly on pupil perceptions of research in the sciences and the scientific method. The questionnaire was a 25-item Likert Scale (1-5) distributed to seven collaborating schools. We received 2634 returns from pupils across key stages 3, 4 and 5. We also asked teachers to complete the questionnaire to reflect how they thought their pupils would respond. We received 54 teacher responses. Statistically significant differences in the responses were identified through a chi-square test on SPSS and the interpretive part of our analysis considers how the term ‘research’ appears in the national curriculum for England and Wales and the three main English exam boards. The main theoretical construct that informs our analysis is Angela Brew’s 4-tier descriptor of perceptions of research (domino, trading, layer, journey). We use this framework to map the national curriculum for Science in England to establish the when, what and how research is presented to school pupils in England and Wales. We highlight and discuss certain pupil views on: research as involving the identification of a research question; research as a means to confirm one’s own opinion; research as involving the generation of new knowledge and the collection of new data, such as interviews and questionnaires as well as laboratory work, field trips and library searches. We also discuss pupils’ statements of confidence in their ability to do research, school experiences of research, perceptions of difficulty and importance of research to future career choice.", "keywords": [ "Research Perception", "Secondary School", "Research-led Teaching", "National Curriculum", "National Curriculum for Science", "Value", "Career", "Science Education", "STEM" ], "content": "Introduction\n\nResearch is a process that occurs in all disciplines and a society’s knowledge economy is reliant on it. The United Kingdom (UK) is very successful in the quantity and quality of science it produces – it is ranked first in field-weighted citation impact. Despite having only 4.1% of the world’s researchers, it accounts for 15.9% of the world’s most highly cited papers (International Comparative Performance of the UK Research Base, 2013). A good example of research benefiting economy is highlighted in a report by the Institute of Food Research, which is funded by the Biological and Biotechnological Science Research Council (BBSRC). The report demonstrates that for every £1 invested in research, £8 is returned to the UK economy (Brookdale Consulting, 2013). To ensure that the UK maintains economic prosperity in the future, future generations need to engage with science, technology, engineering and mathematics (STEM) subjects. Considerable effort is being made to raise the profile of these subjects in secondary schools in order to encourage pupils to take up these subjects at A level and beyond. It has been noted that, as societies become more reliant on science and technology, fewer school-aged children are choosing science and technology as a career path (Donghong & Shunke, 2010). Clearly, this is a concern: as research by the UK science council suggests in 2017 over 58% of jobs will require skills in STEM subjects (http://www.score-education.org/media/3668/report.pdf).\n\nResearch can be defined in many different ways. For example, the Oxford English Dictionary (OED) defines research as “Systematic investigation or inquiry aimed at contributing to knowledge of a theory, topic, etc., by careful consideration, observation, or study of a subject.” (OED Online http://www.oed.com/view/Entry/163432?rskey=RKm0Mc&result=1#eid). Redman & Mory (1923) defined research as “systematised effort to gain new knowledge”. The four UK research councils as part of the Research Excellence Framework (REF) define research as “a process of investigation leading to new insights, effectively shared” (http://www.ref.ac.uk/pubs/2011-02/).\n\nThe concept of a ‘process of investigation’ is embedded within the philosophy of the scientific method. The concept first emerged from Francis Bacon’s ideas of inductive reasoning and was adopted by the Royal Society in the 1660s as a method to promote systematic investigation (Purver, 2013). One definition of the scientific method as a series of discrete steps which could be used for teaching the scientific method in secondary schools originated with Keeslar in 1945 (Keeslar, 1945). Keeslar designed a questionnaire which listed statements to do with elements of the scientific method. This questionnaire was sent to 22 scientists at the University of Michigan, who then ranked/agreed/disagreed with the statements. The responses were analysed by assigning a relative numerical value to each statement on a 200 point scale, by using a formula designed by Keeslar; a series of 12 steps emerged which formed the basis of the schematics that are taught in schools worldwide (McComas, 1998).\n\nHowever, within the scientific research community, the scientific method is applied in different ways and not always in accordance with the rigid, linear investigation schematic that is often portrayed in text books (Figure 1). New researchers learn how to conduct research through participation in scientific studies under the guidance of experienced researchers (McComas, 1998; Sarma, 2014).\n\nIn 2001 Angela Brew conducted a phenomenographic study into how research was experienced by established researchers. Her investigation uncovered four different ways in which research is perceived:\n\n1. Domino variation-where research is viewed as comprising tasks, events, things, activities, problems, techniques, experiments, issues, ideas or questions.\n\n2. Trading variation-where research is seen as product and social phenomenon, e.g. in terms of publications, grants and social networks.\n\n3. Layer variation-where research brings to light ideas, explanations and truths.\n\n4. Journey variation-where the activities in which the researcher engages enables them to grow or transform.\n\nWe note that there are very few studies that have looked at the perceptions of research by secondary school pupils and the value they place on research in relation to their future careers. Studies that have been conducted in this research area have focused on postgraduate students in higher education (Meyer et al., 2005) or experienced researchers (Brew, 2001). We see our project as a potential contributor to this under-researched area by exploring how pupils currently conceive research and science. We ask the following questions:\n\nHow is the term research used in the national curriculum, the national curriculum for science in England and in examination board specifications?\n\nDo pupils consider research to be a process or an output?\n\nDo pupils consider research to be challenging?\n\nDo pupils consider research to be valuable to them for their future?\n\nDo pupils consider that they do research within the school environment?\n\nHow do teachers think pupils perceive research?\n\n\nMaterials and methods\n\nA questionnaire was designed in a series of research team meetings in the early months of the study. Starting from one of the widely-used and reliability-tested Fennema-Sherman Mathematics Attitudes Scales (Fennema & Sherman, 1976; Wikoff & Buchalter, 1986), 25 items were constructed around the four themes who does research, the value of research, the process of research, and myself and research (6, 4, 9 and 6 items respectively). Attention was given to the inclusion of both positive and negative statements. Seven schools located in East Anglia participated (Table 1). The questionnaire was piloted to about 600 pupils in School C and refined further prior to its use, with randomised item order, with the large cohort of about 6,000 pupils. For the questionnaire see the supplementary information. The questionnaire was distributed to and collected from school pupils across all year groups by school teachers. Pupils completed the questionnaire during their morning registration period. We received 2634 responses. The questionnaires were scanned by the data collection company Kendata (http://www.kendata.com/), and an Excel database of responses was compiled and then imported into SPSS version 22. For statistical analysis the year groups were collated according to key stage (Table 2) and the Likert scale was coded in SPSS as strongly agree/agree (1); neither agree not disagree or unsure (2) and disagree/strongly disagree (3). The data was analysed using Pearson’s chi-square test. It was recognised that large data sets can yield small p values; so to increase the robustness of the analysis the probability was set at <0.001 in order to be deemed significant.\n\n1Rating is as determined by the Office for Standards in Education, Children’s Services and Skills (Ofsted).\n\nThe questionnaire was converted into an online form, and teachers were asked to fill it in according to how they thought their pupils would respond. The teacher sample size was 54, with 49 from state schools and 5 from an independent school. In order to compare the pupil and teacher data a randomised stratified sample (n=54) of the complete pupil data set (n=2634) was compared against the data from the teachers. The data was analysed using Pearson’s chi-square test. Due to the smaller sample size [n=108] compared to the total pupil questionnaire data [n=2634] the probability was set at <0.01 in order to be deemed significant.\n\nThe questionnaire on research perception was distributed to the seven schools, and was provided to pupils by form tutors during the morning registration period. There were a total number of 2634 returns, with the sample split in terms of gender and key stage as shown in Table 2.\n\nAll phases of the research have been approved by the School of Education’s Research Ethics Committee (EDU-REC). Consent for participation in the project was secured through the distribution of information sheets and collection of signed consent forms from teachers, parents and pupils over the age of 16. As a complement to parental consent pupils under the age of 16 signed assent forms. Participation in the study took place during school time (either during lessons or outside lessons) and as part of the students’ learning experience about research. The teachers encountered no problems as their schools are official partners of the project and participation implied minimal interference with one normal school day and was carried out with adequate notice. Across all phases of the study, including the analysis of the data and the dissemination of the findings, confidentiality, anonymity and right of withdrawal rules have applied throughout. We note that EDU-REC complies with the British Educational Research Association’s Revised Ethical Guidelines for Educational Research. The research team carried out the research in awareness of the relevant sections of the Data Protection Act (1998): http://www.hmso.gov.uk/acts/acts1998/19980029.htm and Freedom of Information Act (2005).\n\nThe purpose and procedures of the research, and the potential benefits and costs of participating (e.g. the amount of their time involved) were fully explained to teachers, parents and pupils at the outset. The full identity of all members of the research team was revealed to potential participants. No pressure was placed on any individual or institution to participate in the study and the treatment of no individual was in any way prejudiced if they chose not to participate in the project. Schools, teacher and parents were provided with the UEA contact details of team members (not personal contact details) in order that they could make contact in relation to any aspect of the research, should they wish to do so. We notified participants that complaints could be made to the EDU Head of School. Participants were made aware that they may freely withdraw from the project at any time without risk or prejudice. Research activities were carried out with regard for mutually convenient times and negotiated in a way that seeks to minimise disruption to schedules and burdens on teachers, pupils and their parents. The views of all participants in the research were respected. The team was alert and sensitive to any prejudice that may emerge from differences relating to age, culture, disability, race, sex, religion and sexual orientation, when planning, conducting and reporting the research. The original hard copies of the questionnaires and the interview audio recordings and transcripts are kept in a safe and secure location and are being used purely for the purposes of the research project (including dissemination of findings). No-one other than research colleagues have access to any identifiable raw data collected. Participants have been informed that they have the right of access to any data pertaining to them. All necessary steps have been taken to protect the privacy and ensure the anonymity and non-traceability of participants – e.g. by the use of pseudonyms, for both individual and institutional participants, in any written reports of the research and other forms of dissemination.)\n\n\nResults\n\n\n\n\nThe representation of research in the national curriculum for science in England and the examination board specifications\n\nTwo researchers (the first two authors) undertook the mapping of the national curriculum for Science in England (NCSE) for key stage 3 (https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/335174/SECONDARY_national_curriculum_-_Science_220714.pdf), key stage 4 (https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/381380/Science_KS4_PoS_7_November_2014.pdf) and key stage 5 (https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/446829/A_level_science_subject_content.pdf) to Brew’s framework independently. We used the documents pertaining to teaching in the 2013–14 academic year, as this was when the data was collected. Initially, a discussion was held to ensure that both researchers held a shared understanding of each component of the framework. Each individual learning outcome of key stage 3, 4 and 5 was assigned as trading, journey, domino and variation. Assignment of learning outcomes by both researchers was compared and, where disagreement arose, discussion was held with a third researcher until a consensus was reached. The national curriculum (NC, across areas of study) was also scrutinised for mention of the word ‘research’, or any phraseology that could be identified as referring to research (https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/335116/Master_final_national_curriculum_220714.pdf).\n\n\nThe national curriculum and the national curriculum for science in England\n\nAs the research in this paper was conducted in England, references to curriculum are restricted in this region. The NC provides all local authority-maintained schools in England “the programmes of study and attainment targets for all subjects, at all key stages” (p.13) (https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/335116/Master_final_national_curriculum_220714.pdf). The key stages (KS) are described in Table 3 and contain key learning milestones that should be delivered to pupils across the breadth of taught subjects and disciplines. It is acknowledged that independent schools, free schools and academies do not need to follow the NC. The national curriculum for science in England (NCSE) applies to Biology, Chemistry and Physics and at key stage 5 (KS5) it also includes psychology.\n\nAn initial analysis of the NC demonstrates that science is placed in high regard and it is felt to be of importance to society. There is also a desire that pupils appreciate this importance: “Science (..) is vital to the world’s future prosperity and all pupils should be taught essential aspects of the knowledge, methods, processes and uses of science” (p.168). A key aim is pupils should “develop understanding of the nature, processes and methods of science through different types of science enquiries that help them to answer scientific questions about the world around them” (p.168) (https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/335116/Master_final_national_curriculum_220714.pdf). The NCSE has also established a key aim for pupil attainment with a main outcome of the curriculum in key stages 4 and 5 that pupils should appreciate and establish an optimistic and positive view of the role and impact of science in providing solutions to societal problems “pupils should appreciate the achievements of science” (p.3); ‘the role of science in understanding the causes of and solutions to some of the challenges facing society” (p.4) (https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/381380/Science_KS4_PoS_7_November_2014.pdf). The NCSE at key stage 5 provides a strong, positive vision that pupils should acknowledge science as a solution provider on behalf of society. They are required to understand ‘how society makes decisions about scientific issues and ‘how sciences contribute to the success of the economy’ (p.3) (https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/446829/A_level_science_subject_content.pdf).\n\nThe requirements of a future society with a workforce with skills in STEM is stressed in the curriculum. In key stage 4 (KS4) the NCSE states that teaching should “establish the basis for a wide range of careers”(p.3) (https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/381380/Science_KS4_PoS_7_November_2014.pdf). The KS5 documentation lists a key aim to “develop (their) interest in and enthusiasm for the subject, including developing an interest in further study and careers associated with the subject” (p.3) (https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/446829/A_level_science_subject_content.pdf).\n\nThe NCSE was mapped against the Brew (2001) framework and this is shown in Table 4.\n\nThe analysis presented in Table 4 indicates that the NCSE maps to all aspects of the Brew (2001) framework. Thirty seven separate learning outcomes were identified across KS 3, 4 and 5; 68% of them map onto the domino variation; 22% map to layer and 19% to journey, and only 11% map to trading variation (Table 4). The outcomes linked to ‘experimental skills and strategies’ and ‘analysis and evaluation’ are entirely dominated by the domino variation. This is not surprising as this variation describes research as activity, event, problems, technique and experiment. Learning outcomes under the ‘development of scientific thinking’ are more complex, and have examples mapped to layer variation (bringing to light ideas, explanations and truths), trading variation (research as product and social phenomenon) and journey variation (growth and transformation).\n\nThere are some differences across the different key stages. Learning outcomes that map to journey variation are not apparent at KS3, but do appear at KS4 (5 outcomes) and KS5 (7 outcomes). There are two examples of learning outcomes linked to layer variation at KS3, but this increases through KS4 (7 outcomes) to KS5 (8 outcomes). There are no trading variation outcomes at KS3, but there are four outcomes linked to this variation at both KS4 and 5.\n\nSTEM disciplines require and depend upon research skills and the NCSE describes a series of key learning outcomes, which are clearly part of a process of investigation and map to domino variation:\n\n1. Ask questions and make predictions using scientific knowledge.\n\n2. Carry out appropriate scientific enquiries to test predications.\n\n3. Record observations and measurements and apply sampling techniques.\n\n4. Present and interpret observations and data.\n\n5. Present explanations in relation to predictions and hypothesis.\n\n6. Identify further questions arising from results.\n\nWhile the steps of the ‘scientific method’ are referred to within the NC, the actual term ‘scientific method’ is not present. Instead the phrase “working scientifically” is used to describe “the key features of science enquiry, so that pupils learn to answer relevant scientific questions” (p.169) (https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/335116/Master_final_national_curriculum_220714.pdf).\n\nThe word ‘research’ is not used at all in the two documents for KS 3 or 4. The concept promoted by Redman & Mory (1923) where research is defined as the ‘systematised effort to gain new knowledge’ and the definition from the REF with research as “a process of investigation leading to new insights, effectively shared” is not explicitly stated within the NCSE although it is suggested that pupils should “use scientific theories and explanations to develop hypotheses” (p.5) and “interpret observations and other data including identifying patterns and trends, making inferences and drawing conclusions” (p.6) (https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/381380/Science_KS4_PoS_7_November_2014.pdf). Within the KS5 document the word ‘research’ is specifically linked to psychology (rather than to biology, physics or chemistry) students must develop knowledge and “understanding of research in psychology” (p.16) (https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/446829/A_level_science_subject_content.pdf). The word ‘research’ can also be found in Appendix 5 of the key stage 5 NCSE that states practical work undertaken by students throughout the A level syllabus should include ‘research and referencing’. This includes “the use of online and offline research skills including websites, textbooks and other printed scientific sources of information” and “correctly cite sources of information” (p.20) (https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/446829/A_level_science_subject_content.pdf).\n\n\nThe examination board specifications\n\nIndependent schools, free schools and academies do not need to follow the NCSE. However all schools in England offer qualifications through the three major exam boards in England: the Assessment and Qualifications Alliance (AQA), Edexcel (Pearson-London Examiners) and Oxford, Cambridge and Royal Society of Arts & Manufactories Examinations (OCR). These exam boards offer a range of qualifications, including the General Certificate of Secondary Education (GCSE), the Business and Technology Education Council (BTEC) and the General Certificate of Education (GCE). Thus we thought it important to look at the specification of qualifications from different exam boards to see how the term ‘research’ is used within this documentation. We focussed the investigation onto GCSE and GCE qualifications in biology as an example as the schools in this study all offer these courses.\n\nAt GCSE level the pupils are expected to consider evidence from different areas of scientific research, as shown by statements that include “explain how new evidence from DNA research and the emergence of resistant organisms supports Darwin’s theory” (p.20) (http://www1.edexcel.org.uk/science2011/GCSE_Biology.pdf) as well as to think about the “the social and ethical issues concerning the use of stem cells from embryos in medical research and treatments” (p.39) (http://filestore.aqa.org.uk/subjects/AQA-BIOL-W-SP-14.PDF). The OCR specification also clearly links the term ‘research’ to fact-finding, e.g. “research diabetes and how it can be managed’ (p.24) and ‘research the work of John Ray and Carl Linnaeus in developing a modern classification system” (p.30) (http://www.ocr.org.uk/Images/82545-specification.pdf).\n\nIt transpires that in all the examination boards the controlled assessment requires the use of research, but the term is linked to secondary research, which can include extracts from books and websites. Students can carry out secondary research in a library or at home (http://www1.edexcel.org.uk/science2011/GCSE_Biology.pdf). As part of the controlled assessment pupils “plan and carry out an investigation to collect primary data to test their hypothesis” (p.116) (http://www.ocr.org.uk/Images/82545-specification.pdf) but the term ‘research’ is not linked to this activity, but only to the former ‘fact finding’ part of the controlled assessment. Thus in these GCSE specifications investigation and research is split. The actual practical work is termed ‘investigation’, fact finding leading up to this is termed ‘research’.\n\nAt GCE level the term ‘research’ is used for evidence of practical work and as part of practical competency “uses appropriate software and/or tools to process data, carry out research and report findings” (p.38) (http://qualifications.pearson.com/content/dam/pdf/A Level/biology-b/2015/specification-and-sample-assessment-materials/9781446914533_GCE2015_A_BIOLOGYB for web.pdf). As with GCSE it is linked to fact finding “use online and offline research skills including websites, textbooks and other printed scientific sources of information” (p.10) (http://www.ocr.org.uk/Images/171736-specification-accredited-a-level-gce-biology-a-h420.pdf). The OCR specification now has a ‘research skills’ element to their practical portfolio which consists of the following:\n\nApply investigative approaches\n\nUse online and offline research skills\n\nCorrectly cite sources if information\n\nWithin the AQA GCE biology specification the term research is only mentioned under practical mastery, “carry out research and report findings” (p.75) (http://filestore.aqa.org.uk/subjects/specifications/alevel/AQA-2410-W-SP-14.PDF). The information presented here on the use of the term ‘research’ in qualification specification corresponds well to the use of ‘research’ in the NCSE.\n\nThe application of the term research in these different scenarios, on the one hand linking research to cutting edge scientific knowledge ‘embryonic stem cells’ but also linking it to basic ‘fact-finding’ at GCSE and GCE leads to a confusion over what research really is, which is evident in this paper.\n\nWe now present the questionnaire data on pupil perceptions of: what constitutes research; their experience and ability in research; and, their appreciation for research.\n\n\nThe questionnaire data on pupil perceptions of research\n\nA fundamental part of the research process is the establishment of the research question. The NCSE at key stage 3 clearly indicates that students are expected to “ask questions and develop a line of enquiry based on observation and prior knowledge and experience” (p.4) (https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/335174/SECONDARY_national_curriculum_-_Science_220714.pdf). To explore pupils’ perceptions of using questions within research and science investigation, Figure 2 shows the responses to the statement ‘research always involves investigating a question’. The response indicates that pupils were unclear that research should begin this way, only 38.8% strongly agreed or agreed with the statement. There was no significant difference in responses with regard to either gender or KS (χ2[2, N=2362] 12.26, p=0.002) and (χ2[4, N=2585] 16.80, p=0.002) respectively. This suggests that the perception of the importance of posing research questions did not increase as students gained more science investigation experience through their education. When teachers were asked how their pupils would respond to the statement there was no statistical difference in how the pupils responded and how the teachers thought they would respond, (χ2[2, N=108] 4.54, p=0.1). The NCSE clearly indicates there is a requirement for pupils in secondary education to “ask questions” in relation to scientific investigation (p.4) (https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/335174/SECONDARY_national_curriculum_-_Science_220714.pdf). The data presented here potentially indicates an issue with how this aspect of scientific inquiry and scientific process occurs in the school environment.\n\nThe scientific method requires the researcher to minimise bias. In addition the NCSE indicates that pupils should “pay attention to objectivity and concern for accuracy” (p.4). Figure 2 shows that substantial number of pupils (50%) strongly agreed or agreed that you do research to confirm your own opinion. There was no significant difference in responses according to either gender or KS (χ2[2, N=2355] 6.40, p=0.04) and (χ2[4, N=2576] 4.78 p=0.3) respectively, indicating that this does not change with increasing research experience. There was also no statistical difference in how the pupils responded and how the teachers thought they would respond, (χ2[2, N=108] 0.63, p=0.73).\n\nRedman & Mory (1923) define research as ‘systematised effort to gain new knowledge’. In addition the NCSE suggests that pupils should “use scientific theories and explanations to develop hypotheses” (p.5) and “interpret observations and other data including identifying patterns and trends, making inferences and drawing conclusions” (p.6) (https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/381380/Science_KS4_PoS_7_November_2014.pdf). However no clear learning outcome is provided that asks pupils to understand that scientific inquiry or research is a systemised effort to gain new knowledge. When we investigated pupils understanding of this with ‘the main purpose of research is to generate new knowledge’ more than 70% of pupils across all key stages strongly agreed/agreed with the statement. There was no significant difference is responses across KS (χ2[4, N=2577] 4.18, p=0.43) or gender (χ2[2, N=2356] 14.7, p=0.001). There was also no statistical difference in how the pupils responded and how the teachers thought they would respond, (χ2[2, N=108] 2.26, p=0.32).\n\nPupils were asked if research involves collecting new data (Figure 3). There was no significant difference in the way in which males and females responded to the statement ‘research involves collecting new data’ (χ2[2, N=2356] 14.1, p=0.001). However, there was a significant difference across KS (χ2[4, N=2577] 22.16, p<0.001) with more pupils from KS3 (76.7%) strongly agreeing/agreeing with this statement than KS4 (69.2%) or 5 (67.5%). This could reflect a greater understanding at KS5 of how existing research data can be combined together and re-used in meta-analysis. This suggests a more sophisticated view of research which grows with experience. Overall though, pupils were more likely to strongly agree/agree with this statement (71.2%). There was no statistical difference in how the pupils responded and how the teachers though they would respond, (χ2[2, N=108] 0.30, p=0.86).\n\nThe NCSE states that school pupils should “select, plan and undertake appropriate types of scientific enquiry to test predictions including the use of variables and use appropriate techniques, apparatus and materials in field and lab work including issues of health and safety” (p.4) as well as “make and record observations and measurements using a range of methods” (p.4) (https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/335174/SECONDARY_national_curriculum_-_Science_220714.pdf). In Figure 4 pupils show an understanding that research could be conducted in areas other than a laboratory. There was no significant difference in the way in which males and females responded to the statement ‘research is carried out solely through experiments in a laboratory’ (χ2[2, N=2360] 7.74, p=0.02). There was however a significant difference across KS (χ2[4, N=2581] 124.97, p<0.001) with more pupils from KS5 (77.9%) disagreeing/strongly disagreeing with this statement than KS4 (67.5%) or KS3 (52.9%), again hinting at the greater experience of research methods and techniques as pupils move through the key stages. There was no statistical difference in how the pupils responded and how the teachers thought they would respond, (χ2[2, N=108] 0.37, p=0.83).\n\nWhen asked to respond to the statement ‘research can be carried out through collecting data during a field trip’, there was no difference in response according to gender or across KS (χ2[2, N=2368] 8.91, p=0.01) and (χ2[4, N=2590] 10.85, p=0.03) respectively (Figure 5). The majority of respondents strongly agree or agreed with this statement (82.1%). There was no statistical difference in how the pupils responded and how the teachers thought they would respond, (χ2[2, N=108] 1.29, p=0.53).\n\nThis was an almost identical response for the statement ‘research involves searching through sources such as libraries’ (Figure 5) with no significant difference in response according to gender or KS (χ2[2, N=2349] 8.23, p=0.02) and (χ2[4, N=2538] 3.40, p=0.69) respectively (Figure 4). The majority of respondents strongly agreed/agreed with this statement (81.5%). There was no statistical difference in how the pupils responded and how the teachers thought they would respond, (χ2[2, N=108] 3.43, p=0.18).\n\nFigure 6 shows that pupils clearly understand that research can involve collecting data through interviews and questionnaires. There was no significant difference in the way in which males and females responded to the statement ‘research can involve collecting data through interviews and questionnaires’ (χ2[2, N=2368] 6.33, p=0.42). There was however a significant difference in the respondents across KS (χ2[4, N=2590] 53.46, p<0.001). Pupils in KS5 (92.7%) are more likely to strongly agree/agree than KS4 (84.7%) and KS3 (80.9%) pupils. The majority of respondents strongly agreed/agreed with this statement (86.2%). This reflects the greater experience of research of KS5 pupils. There was no statistical difference in how the pupils responded and how the teachers thought they would respond, (χ2[2, N=108] 2.61, p=0.27).\n\n\nThe questionnaire data on pupil confidence in their research experience and ability\n\nPupils are confident that they do research, and they think they do it in their school environment (Figure 7a). For the statement ‘I am confident that I can do research’ there was no significant difference in response according to gender or across KS, (χ2[2, N=2373] 4.89, p=0.09) and (χ2[4, N=2593] 3.93, p=0.41) respectively. The majority of respondents (82.5%) strongly agreed/agreed with this statement. There was also no significant difference according to gender or across KS in responses to the statement ‘I think I do research in school’, (χ2[2, N=2586] 9.88, p=0.007) and (χ2[4, N=2586] 7.57, p=0.1) respectively (Figure 7a). The majority of pupils (83.4%) strongly agreed/agreed with this statement.\n\n(a) Percentage of pupil responses to the ‘I am confident that I can do research’ and ‘I think I do research in school’. (b) Comparison of the percentage distribution of responses from pupils and teachers to the statement ‘I am confident that I can do research’.\n\nThere was however a significant difference in how the teachers thought pupils would answer this question (χ2[2, N=108] 14.37, p=0.001). The teachers thought the pupils would be much less confident that they could do research (Figure 7b). There was no significant difference between pupils and teachers on responses to the statement ‘I think I do research in school’ (χ2[2, N=108] 1.40, p=0.49).\n\nAs pupils progress through their educational experience, it is assumed that the work they are asked to do which involves research becomes more and more challenging. When asked to rate the statement ‘doing research is challenging’, there was a significant difference in the way in which pupils across KS responded (Figure 8a). Pupils in KS5 were more likely to strongly agree/agree with this statement than those in either KS3 or 4. (χ2[4, N=2589] 72.49, p<0.001). However, despite the assumed increase in challenging work, there was no significant difference between KS3 and 4 (χ2[2, N=1748] 4.29, p=0.1). There was also no difference in how males and females responded to this statement (χ2[2, N=2367] 1.94, p=0.38). There was however a significant difference in how teachers and pupils responded to this statement (χ2[2, N=108] 13.25, p=0.001), with teachers thinking that pupils would find research challenging (Figure 8b).\n\n(a) Percentage pupil responses across key stage to the statement ‘doing research is challenging’. (b) Comparison of the percentage distribution of responses from pupils and teachers to the statement ‘doing research is challenging’.\n\n\nThe questionnaire data on pupil appreciation for research\n\nIn order for the UK to benefit in the future from a knowledge economy, pupils currently in school need to value research and think it of value to their careers. There was a significant difference in how pupils across KS responded to the statement ‘research is a worthwhile activity’ (Figure 9) (χ2[4, N=2589] 72.99, p<0.001). Pupils in KS5 were more likely to strongly agree/agree with this statement than KS3 (χ2[2, N=1759] 72.92, p<0.001) or KS4 pupils (χ2[2, N=1671] 48.70, p<0.001). There was also a significant difference in how KS3 versus KS4 pupils (χ2[2, N=1748] 22.93, p<0.001) responded. This shows that as pupils progress through their education, they value research more. There was no significant difference in how males and females responded (χ2[2, N=2370] 10.18, p=0.006).\n\nThere was no statistical difference in how the pupils responded and how the teachers thought they would respond, (χ2[2, N=108] 0.43, p=0.81).\n\nThere was no significant difference in responses according to gender or across KS to the statement ‘knowing how to do research will help me in my future career (χ2[2, N=2363] 6.59, p=0.04) and (χ2[4, N=2584] 9.19, p=0.06) respectively (Figure 10). The majority of respondents strongly agreed/agreed with this statement (76.9%). However, there was a significant difference in how teachers thought pupils would respond, with teachers thinking that pupils would not respond positively to this statement (χ2[2, N=108] 27.57, p<0.001).\n\n\nDiscussion\n\nAs stated in the introduction research can be defined in many different ways. Two of these three definitions include the word ‘new’ (Redman & Mory, 1923; Research Excellence Framework http://www.ref.ac.uk/pubs/2011-02/), and this poses the following question: is research only about finding out new and original knowledge which is not known to anyone? Or can it also be applied to new knowledge not previously known to self, but known to others? The former is clearly the case for the REF where new, original research is judged. However, the latter scenario is often the case in schools, for example, where pupils are asked for homework to do ‘research’ in a particular area. This type of research is content driven ‘fact’ finding, the research question, or even just the topic often having been given as part of the homework task. Leedy & Ormrod (2010) describe this as ‘information discovery’ and do not consider it to be research. In terms of formal education, the NCSE only uses the term ‘research’ when linked to finding facts and using secondary sources. This is also clearly the case in exam board specifications, where pupils are required to conduct secondary research as part of controlled assessments. Thus the word ‘research’ can be applied to different scenarios of fact finding and data comparison. We feel that research as either new to ‘self’ or ‘new to all’ and thus original, is a crucial distinction in meaning, and colours how the term ‘research’ is both perceived and used by different groups of people, e.g. school pupils, teachers, government bodies, exam organisations, universities, as well as novice and experienced researchers. This is important because as pupils transition through their educational career the meaning and use of the word ‘research’ changes. One example of this is in higher education (HE) where leading universities are keen to promote their research-led teaching manifesto, where teaching is informed by research and research activity goes beyond fact finding and the investigation of secondary sources, and into novel enquiry and original investigation (Yeoman & Zamorski, 2008).\n\nDespite the specific linking of research in the NC to fact finding, and the lack of the use of the word ‘research’ in the NCSE at KS3 and 4, the learning outcomes of the NCSE do map onto the different research variations as outlined by Brew (2001). The mapping is dominated by domino variation, that sees research categorised as task, activity, event, problems, technique and experiment. These scientific capabilities in pupils are important when demonstrating ‘scientific mastery’ as required by examination boards.\n\nWhile the steps of the ‘scientific method’ are referred to within the NC the actual term ‘scientific method’ is not present. Instead the phrase ‘working scientifically’ is used to describe “the key features of science enquiry, so that pupils learn to answer relevant scientific questions” (p.169) (https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/335116/Master_final_national_curriculum_220714.pdf.). Whilst many scientists struggle to recognise the step-by-step scientific method as portrayed by Keeslar (1945) in how they conduct their research, what is not in contention is that research must begin with a sensible question. This question can arise from ongoing observation and experimentation, or it might come from a systematic review of existing research. Despite the fact that “asking questions” appears in the NCSE, Reiss (2015) states that “we don’t do a very good job of getting pupils in school science lessons to ask the sorts of questions that scientists actually ask”. This study provides evidence that less than 40% of secondary school pupils thought that it was necessary to start research with a question. In order to ascertain if a research question is worth pursuing, then background information must be gathered to see if answers to the question already exist, or if the question needs to be refined in the context of what is already known. However, in the school environment, the search for background information is often divorced from the actual question setting thus the whole picture of scientific enquiry cannot emerge. Initiatives such as the Extended Project Qualification (EPQ) AS- level and the new GCSE equivalent, will help with this issue, and allow pupils to experience full scientific enquiry. The EPQ is a dissertation or investigation/field study which involves establishing and then addressing a research question through either a literature review and argumentative discussion or data collection and analysis. In the 2014–15 academic year 33,564 pupils completed the EPQ (http://www.jcq.org.uk/examination-results/a-levels/2015/a-as-and-aea-results-summer-2015). As Malcolm Trobe (Deputy General Secretary of the Association of School and College Leaders) stated in a recent BBC article (http://www.bbc.co.uk/news/education-33819871).\n\n\"(EPQs) are phenomenally valuable in giving young people the opportunity to prepare themselves for university where they will spend much of their time studying and learning through their own research and reading.\"\n\nOne of the other issues we raised in this paper is that of being unbiased during systematic investigation. Only 16.8% of pupils disagreed/strongly disagreed, with the statement ‘you do research to confirm your own opinion’. One of the core premises of the scientific method is that researchers remain unbiased and the NCSE at all key stages requires pupils to “pay attention to objectivity” (p.4) (https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/335174/SECONDARY_national_curriculum_-_Science_220714.pdf). Confirmation bias is a well-known phenomenon and it is where researchers (including scientists) tend to look for and only see evidence that confirms what they already believe (Nickerson, 1998).\n\nOnly 32% of pupils found research challenging at KS3 and 4, but this increased to 49% at KS5. This reflects the increased complexity of the material taught at A level, the requirement for more critical analysis of sources, the chance to do more sophisticated practicals and fieldwork, as well as the opportunity to do qualifications such as the EPQ (Level 3). It is perhaps surprising not to see an increase in percentage between KS3 and 4, suggesting that teachers could challenge pupils more at KS4 in terms of scientific enquiry. This could be resolved by the introduction of Level 2 project qualification such as that offered by the AQA exam board (http://www.aqa.org.uk/subjects/projects/aqa-certificate/PQ2-7992/spec-at-a-glance). Another interesting finding linked to this is that teachers think that pupils will find research more challenging than perhaps they do. Again this may come down to the perception of the term research. The majority of teachers are graduates, with HE research experience; when teachers set pupils homework tasks to ‘research’ a topic, they may be using the word ‘research’ in a different way to how they would actually define it, thus leading to the disparity seen in this study.\n\nThe UK has a knowledge economy dependent upon science and research. Thus we have a need for STEM subjects to be taught in schools and to encourage the new workforce to take STEM subjects to match STEM need in the future. This pipeline requires pupils to understand the range of careers which require STEM subjects. As part of the Education Act of 2011, the government placed responsibility for career guidance into individual schools, rather than it being provided by local authorities or central government. However, there was no funding and no guidance on how this should be achieved (Hooley et al., 2012). In a review, ‘Career 2020’, jointly written by the Pearson Think Tank and International Centre for Guidance Studies at the University of Derby, Hooley et al. recommend that we encourage schools to think of careers as being “a key component of their mission and to actively link this to the curriculum” (p.4) (http://derby.openrepository.com/derby/bitstream/10545/251032/1/CAREERS+2020.pdf). There is evidence which suggests that this approach of linking careers to the curriculum is the most effective, but requires considerable buy-in from school senior leadership teams. It is stated in the KS5 NCSE that pupils should “develop an interest in further study and careers associated with the subject” (p.3) (https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/446829/A_level_science_subject_content.pdf), this outcome however is missing in the NCSE for KS3, and only briefly mentioned at KS4 where is states that teaching should “establish the basis for a wide range of careers” (p.3) (https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/381380/Science_KS4_PoS_7_November_2014.pdf).\n\nThis research presented here suggests that pupils think that research will be valuable to them in their future career, although it was also clear that teachers did not think that pupils would value this as much as they did. As discussed earlier this may be due to the perception of the term ‘research’. Pupils also think research is a worthwhile activity, and this positive feeling increases during their educational career, possibly as they are exposed to more opportunity, e.g. through the EPQ. These positive views are examples of how research is seen as ‘journey’ where activity enables growth and transformation within the Brew (2001) framework. We are now seeking more nuanced and elaborate pupil perceptions through the analysis of focus group interviews that we conducted after the questionnaire.\n\nFinally, Brew (2001) suggests that the framework would be a useful tool to evaluate research performance by individuals, but we have also found that it provides a framework to map curricula.\n\n\nData availability\n\nF1000Research: Dataset 1. Complete pupil data set, 10.5256/f1000research.7449.d108247 (Yeoman et al., 2015a).\n\nF1000Research: Dataset 2. Compiled teacher: pupil data set, 10.5256/f1000research.7449.d108248 (Yeoman et al., 2015b).", "appendix": "Author contributions\n\n\n\nKY and EN designed the pilot questionnaire. KY trialled its use in our lead school and analysed the initial data. KY, EN and LB then amended the questionnaire. KY distributed the questionnaire to the schools involved and collected the data. KY analysed the data. KY and LB independently mapped the national curriculum. KY, LB and EN all prepared the draft manuscript. KY, LB and EN all had significant academic input to the preparation of the paper.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe Research Councils UK (RCUK) provided funding to twelve projects across the UK as part of a funding scheme ‘the school: university partnership initiative’ (SUPI) (http://www.rcuk.ac.uk/pe/PartnershipsInitiative/). This project is part of the Public Engagement with Research Catalyst Scheme with a main objective to support the STEM agenda. The University of East Anglia (UEA) received one of these twelve awards (EP/K027980/1).\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\nWe thank our partner schools for participating in this study.\n\n\nSupplementary material\n\nQuestionnaire on secondary school pupils’ perceptions of research.\n\n\nReferences\n\nBrew A: Conceptions of Research: a phenomenographic study. Stud High Educ. 2001; 26(3): 271–285. Publisher Full Text\n\nBrookdale Consulting:. Impact of the Institute of Food Research. 2013. Reference Source\n\nDonghong C, Shunke S: The more, the earlier, the better: science communication supports science education. editors. Cheng D, Claessens M, Gascoigne T. Springer; 2010.\n\nFennema E, Sherman JA: Fennema-Sherman Mathematics Attitudes Scales: Instruments designed to measure attitudes toward the learning of mathematics by females and males. J Res Math Educ. 1976; 7(5): 324–326. Publisher Full Text\n\nHooley T, Marriot J, Watts AG, et al.: Careers 2020: Options for future careers work in English schools. Pearson. 2012. Reference Source\n\nInternational Comparative Performance of the UK Research Base. In: Department of Business IaS. editor. Elsevier; 2013. Reference Source\n\nKeeslar O: The elements of scientific method. Sci Educ. 1945; 29(5): 273–8. Publisher Full Text\n\nLeedy PD, Ormrod JE: Practical Research: Planning and Design. Ninth Edition, published by Merrill. Pearson Education, Inc. 2010. Reference Source\n\nMcComas WF: The Nature of Science in Science Education: Rationales and Strategies. Kluwer Academic Publishers; 1998; 53–70.\n\nMeyer JH, Shanahan MP, Laugksch RC: Students’ Conceptions of Research. I: A qualitative and quantitative analysis. Scand J Educ Res. 2005; 49(3): 225–244. Publisher Full Text\n\nNickerson RS: Confirmation Bias: A Ubiquitous Phenomenon in many guises. Rev Gen Psychol. 1998; 2(2): 175–220. Reference Source\n\nPurver M: The Royal Society: Concept and Creation, Routledge Library Editions: History and Philosophy of Science. Routledge, 2013; 25. .\n\nRedman LV, Mory AVH: The Romance of Research. The Williams and Wilkins Company; 1923.\n\nReiss MJ: The Nature of Science. In: Learning to Teach Science in the Secondary School: A Companion to school experience. editor, Toplis R, 2015; 64–73. Reference Source\n\nSarma GP: The Art of Memory and the Growth of the Scientific Method. Cornell University Library, arXiv: 1307.0254 (physics.hist-ph). 2014. Reference Source\n\nThe Oxford English Dictionary. \"research, n.\" OED online, Oxford University Press. Reference Source\n\nWikoff RL, Buchalter BD: Factor analysis of four Fennema-Sherman mathematics attitude scales. Int J Math Educ Sci Technol. 1986; 17(6): 703–706. Publisher Full Text\n\nYeoman KH, Zamorski B: Investigating the impact on skill development of an undergraduate scientific research skills course. beej. 2008; 11: 5. Reference Source\n\nYeoman K, Bowater L, Nardi E: Dataset 1 in: The representation of research in the National Curriculum and secondary school pupils’ perceptions of research, its function, usefulness and value to their lives. F1000Research. 2015a. Data Source\n\nYeoman K, Bowater L, Nardi E: Dataset 2 in: The representation of research in the National Curriculum and secondary school pupils’ perceptions of research, its function, usefulness and value to their lives. F1000Research. 2015b. Data Source" }	[ { "id": "11568", "date": "21 Dec 2015", "name": "Beatrix Fahnert", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSupporting the development of a realistic perception of research by pupils is crucial in our aims to inspire the next generations of scientists, and to scientifically engage well-informed next generations of the public at a level necessary for the benefit of everyone (e.g. in context of economy, healthy lifestyle, openness towards technology, sustainability) and having been enabled to evaluate relevant pros and cons when making decisions. Therefore, it is necessary to understand current perceptions, how they arise, how they may change and how they relate to the pupils' engagement with research. An analysis of how the concept of research is embedded in the curriculum, whether research is portrayed as valuable and accessible, and how the educational environment affects pupils' perceptions of research allows to constructively suggest ways forward in the quest.The authors shall be thanked for this important contribution to the field. The aims of the research are clear and well supported by methodology (fully ethically cleared). The questionnaires were piloted and optimised before the large survey.The Abstract summarises the article well.The Introduction sets the scene very well, and the Materials and Methods section was mostly clear including the parameters of the institutions.The very interesting findings are well presented in the Results section. For instance, Table 4 provides a clear mapping of learning outcomes at key stages against the used framework. It visualises how perception and understanding of research should progress through key stages. This then is found in the questionnaire data (e.g. pupils value research more as they progress through education).How research is referred to/not referred to in the curriculum and by exam boards is an interesting finding. The authors also emphasise issues around the use of the term ‘research’ (fact finding vs. novel enquiry) and related consequences in context of e.g. preparing pupils for transition into HE, pupils' understanding of the notion of research-led teaching when selecting their future HEI.A further issue highlighted by the authors is the need for pupils to better understand that research should be unbiased, that a research question is the starting point and how a sound research question is arrived at (and how e.g. the new Extended Project Qualification may support an improved understanding).Teachers were found to mostly correctly gauge their pupils' perception except for whether pupils feel they do research in school, whether pupils find research challenging and whether research is seen as useful by pupils for their future career (importantly here: pupils indeed see the benefit).An encouraging (women in STEM context) finding is that there is no difference in perception between genders. I would like to approve the article and to suggest some minor changes to be made in a revision of the current manuscript.The authors have already provided a contextual discussion of their findings, but have not yet discussed the potential impact of the responders on the data or explained how such impact may have been limited. This needs to be added, because responses are likely provided by a self-selecting group (e.g. 2634 from 6000 pupils; teachers [does the subject background matter?]; institutions already being part of the project). There is also a statement that the study is part of the students’ learning experience about research. How might this have affected responses? I think it would add further value to the figures and make them more self-explanatory if n-values were added as well as a visual indication if there are significant differences in responses; and adding figure legends that state related findings such as response trends (without the actual data, which remain reported in the text as they already are) regarding gender, KS, teacher responses. Some general indication of how 'agree' and 'disagree' responses compare with the reported 'strongly agree/ disagree'/'unsure' would be useful. There are two further points regarding the Materials and Methods section, which the authors may also wish to address when making changes to the current version of the manuscript.The words \"and the interview audio recordings and transcripts\" could be removed to avoid confusion, because these are unrelated to the reported analysis and only relate to subsequent work. The section \"The representation of research in the national curriculum for science in England and the examination board specifications covering the mapping of the national curriculum\" could be part of Materials and Methods rather than Results.", "responses": [ { "c_id": "1772", "date": "19 Jan 2016", "name": "Kay Yeoman", "role": "Author Response", "response": "Thank you for the positive comments and suggestions for improvement. We will be revising and then submitting a new version in the light of your review." }, { "c_id": "1789", "date": "12 Feb 2016", "name": "Kay Yeoman", "role": "Author Response", "response": "Thank you for your useful comments on our paper. We have taken your points into consideration in the revised manuscript and it has improved as a result. The authors have already provided a contextual discussion of their findings, but have not yet discussed the potential impact of the responders on the data or explained how such impact may have been limited. This needs to be added, because responses are likely provided by a self-selecting group (e.g. 2634 from 6000 pupils; teachers [does the subject background matter?]; institutions already being part of the project). There is also a statement that the study is part of the students’ learning experience about research. How might this have affected responses?Response: We have clarified some of these points in responses to the other referee’s comments and including some more clarification in the text. The schools taking part in the project were our partner schools from the RCUK School:University Partnership Initiative (SUPI). A range of schools were chosen for the SUPI on their existing contact with the University and being the type of school which reflected those in Norfolk (e.g. city, rural, coastal). Pupils were not self-selecting on answering the questionnaire, we were dependent on the form teachers conducting the questionnaire during form time and then handing the completed questionnaires back to the link teacher. The teachers responding to the questionnaire would have been from a variety of subject disciplines across the science and the humanities. Yes, this study and the SUPI project is about embedding research activity into the school environment. The questionnaire was conducted as a baseline before the SUPI activities had really been established within the schools.I think it would add further value to the figures and make them more self-explanatory if n-values were added as well as a visual indication if there are significant differences in responses; and adding figure legends that state related findings such as response trends (without the actual data, which remain reported in the text as they already are) regarding gender, KS, teacher responses. Response: We totally agree, and this has been added to the Figure legends. We have included more information in the figure legends. As well as the n= we also included the p value, but the detailed statistic reporting is still in the text.Some general indication of how 'agree' and 'disagree' responses compare with the reported 'strongly agree/ disagree'/'unsure' would be useful.Response: Likert scales are subject to distortion; respondents may avoid extreme responses. The data set presented here is large, and yes there were some differences in the responses to statements between strongly agree/agree as well as strongly disagree/disagree. For example more pupils at KS5 (28.4%) strongly agreed that research is worthwhile activity than either KS3 (19.2%) or 4 (19.1%), but this outcome of a higher percentage of KS5 pupils agreeing with this statement did not change post consolidation of the strongly/agree and agree responses. After consolidation there was a significant difference between KS3 and 4, not apparent when just looking at the strongly agree data alone. This was because far more KS4 pupils (52%) agreed with this statement that KS3 pupils (42%). In our view the consolidation of strongly agree/agree as well as strongly disagree/disagree allowed for a more straightforward statistical analysis using the Chi Squared test and a clearer reporting of the findings. However we do take the point that we may be missing more nuanced findings within the data set by taking this strategy. There are two further points regarding the Materials and Methods section, which the authors may also wish to address when making changes to the current version of the manuscript.The words \"and the interview audio recordings and transcripts\" could be removed to avoid confusion, because these are unrelated to the reported analysis and only relate to subsequent work. Response: We agree and this has been deletedThe section \"The representation of research in the national curriculum for science in England and the examination board specifications covering the mapping of the national curriculum\" could be part of Materials and Methods rather than Results.Response: It is interesting that you mentioned this, as we considered having it there, but decided against as we felt that this was original analysis and a ‘result’ rather than a method." } ] }, { "id": "11953", "date": "18 Jan 2016", "name": "Joanna Verran", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 our opinion, this is a very useful output. The mapping of student and teacher responses against the representation of research within the English national curriculum provides a robust comparison, and the description of the perceptions of students as to the meaning of research is really interesting.The title might better describe the study if it specifically says ‘scientific’ research instead of research. Whilst the authors discuss the other uses of the word research, the questions and answers are specifically in relation to scientific research. We do not particularly like ‘it’s’ in the first line of the abstract. We would also like to see a more clear concluding sentence in the abstract.The study is thorough and the number of participants impressive. It would be interesting to note if all students were taking science, particularly at KS5. We assume they were. Were all the teachers Science teachers? Perhaps a little information within the introduction, as to what other sections of society think of research, might be useful. What do non-scientist adults think for example? There are some interesting reports on this that may help the reader understand the results within the context of society. The mapped curriculum using the Brew descriptors is very interesting. However, the results need more discussion: currently there is very little to describe what they mean, their context, and what, if any, conclusions can be drawn from that analysis. Can the authors evidence their assumption that students have a greater understanding of meta-analysis of data by KS5 than earlier on in their school journey? Or is it a speculative comment. This could be made clearer. How do the findings about the practice of ‘research’ in schools relate to the ‘nature of science’ debate? We also wonder how findings from this study would compare to findings using undergraduates at different stages in their degrees?", "responses": [ { "c_id": "1773", "date": "19 Jan 2016", "name": "Kay Yeoman", "role": "Author Response", "response": "Thank you for the positive comments and suggestions for improvement. We will be revising and then submitting a new version in the light of your review." } ] } ]	1	https://f1000research.com/articles/4-1442
https://f1000research.com/articles/5-164/v1	12 Feb 16	{ "type": "Case Report", "title": "Case Report: Elevated CPK, an indicator of idiopathic inflammatory myopathy?", "authors": [ "Hina N. Khan", "Usman Jilani", "Shitij Arora", "Usman Jilani", "Shitij Arora" ], "abstract": "Polymyositis is a rare disease with incidence rates at about 1 per 100,000 people annually. In this case report we will review a case of proximal muscle weakness with an elevated creatine phosphokinase that was initially misdiagnosed twice as rhabdomyolysis. Therefore, emphasizing that idiopathic inflammatory myopathy is a potential cause of myasthenia that must be considered in the differential. The case will also describe the current treatment and treatment response in polymyositis.", "keywords": [ "Idiopathic inflammatory myopathy", "Polymyositis", "Myasthenia" ], "content": "Introduction\n\nWhen a clinician is confronted with a case of muscle weakness in the setting of a severely elevated creatine phosphokinase by probability alone, the most likely culprit is rhabdomyolysis. Incidence rates of rhabdomyolysis are about four to five-fold that of polymyositis1. This is reflected in the abundance of medical literature citing cases of rhabdomyolysis. This case report highlights a patient who presented with symptoms of an elevated creatine kinase and myasthenia, but was eventually diagnosed with an idiopathic inflammatory myopathy. Idiopathic inflammatory myopathies have only rarely been described in patients as the etiology of these diseases is poorly understood. Mechanisms of action including cellular-mediated cytoxic mechanisms in polymyositis, to a complement-mediated vasculopathy of the small vessels in muscle tissues in dermatomyositis, to a primarily macrophage driven degeneration in immune-mediated necrotizing myopathy have all been ascribed as the pathophysiology behind idiopathic inflammatory myopathies2. Currently, there is an understanding that both genetic and environmental factors play a role in unmasking an inflammatory myopathy3. Our case reviews why the presence of proximal muscle weakness, an elevated creatinine phosphokinase, and systemic clues should raise the clinician’s suspicion for these poorly understand idiopathic inflammatory myopathies.\n\n\nCase\n\nA 64-year-old man presented with two weeks of progressive proximal muscle weakness causing him difficulty ambulating, combing his hair, and raising himself from a seated position. He also reported dysphagia to solids over the past week. The patient was recently admitted twice to an outside hospital for an elevated creatine phosphokinase, and diagnosed with “rhabdomyolysis.” Past medical history was significant for poorly controlled diabetes mellitus. On exam, vitals were within normal limits. Cardiovascular exam revealed regular rate and rhythm with no extra heart sounds and lung exam revealed clear lungs on auscultation. On neurologic exam, patient was awake, alert, and oriented. Cranial nerves II through XII were grossly intact. There was proximal muscle weakness to 4/5 bilaterally in the shoulders and hips, but preserved strength distally. Sensation to light touch remained intact. Hemoglobin was 10.5 g/dL with a normal MCV, WBC were 3.8 K/uL, and platelets were 246 K/uL. Basic metabolic panel revealed a sodium of 141 mEq/L, a potassium of 4.8 mEq/L, and a creatine of 0.90 mg/dl. Transaminases including AST and ALT were 65 and 36 U/L, respectively. CPK was 2705 U/L. ESR and CRP were within normal limits and RF, anti-Ro/La, anti-Jo1, anti-RNP, anti-centromere, anti-Scl-70, and anti-dsDNA were all negative. An MRI of the right arm revealed prominent edema involving the deltoids and an MRI of the bilateral thighs revealed myositis with no discrete collections. An EMG showed evidence of irritative myopathy affecting the bilateral deltoids. A muscle biopsy of the right deltoid was negative for inflammation but presumed to not be representative. He was started on oral Prednisone 60mg daily with resolution of symptoms including weakness and pain and normalization of creatine kinase within one month of starting therapy.\n\n\nDiscussion\n\nMyasthenia, or simply muscle weakness, is a common admitting diagnosis in the inpatient setting with a broad differential. However, in the setting of an elevated CPK the differential diagnosis for myasthenia is narrowed to rhabdomyolysis or a myopathy. While acute exertional rhabdomyolysis can be diagnosed by history, the syndrome of idiopathic inflammatory myopathies must be differentiated from myopathies caused by infections, toxins, paraneoplastic syndrome, and endocrinopathies. Idiopathic inflammatory myopathy encompasses systemic rheumatic diseases including polymyositis and dermatomyositis. Our patient met three out of the four criteria for polymyositis per the American College of Rheumatology including symmetric proximal muscle weakness, elevation of skeletal muscle enzymes, and an abnormal EMG showing polyphasic, short, small motor unit potentials, fibrillation potentials, positive sharp waves, and repetitive high-frequency discharges, suggesting a probable diagnosis of polymyositis. The final criteria needed for definitive diagnosis is an abnormal muscle biopsy with histopathologic findings of degeneration, regeneration, necrosis, and interstitial mononuclear infiltrates, in this case, muscle biopsy was presumed not representative as his anterior right deltoid had been sampled as opposed to the posterior or lateral deltoid which is typically higher yield. However, several epidemiologic features make this a particularly difficult diagnosis in this patient. Foremost, polymyositis is a rare disease with incidence rates occurring in about 1 per 100,000 people annually4–7. Additionally, polymyositis is seen twice as commonly in women than in men. Furthermore, although polymyositis can occur at any age, it typically peaks in the 30–50 year age range7,8, and our patient had a later age of onset. This case also highlights the low sensitivity of serologic tests including anti-Jo-1, anti-Scl-70, and anti-RNP which are prevalent in 21%, 6%, and 5% of patients with polymyositis, respectively9.\n\nHowever, when considering a case of myasthenia with an elevated CPK, some key features can be used to differentiate polymyositis from other diagnoses. These include chronicity of symptoms, presentation of symmetric proximal muscle weakness, and presence of bulbar features, which can be seen in polymyositis, dermatomyositis, or inclusion body disease. Other systemic signs including rash seen in dermatomyositis, or Raynaud Phenomenon seen in scleroderma or CREST can also aid in correctly arriving at a diagnosis. Steroids remain the cornerstone of treatment in polymyositis although lack of response as high as 40% has been reported. Such a high non-response rate may partly be due to misdiagnosis of inclusion body myositis as polymyositis. Studies are ongoing to identify specific mechanisms for steroid resistance with higher expression of proinflammatory molecule “granulysin” in CD8+ T cells and glucocorticoid receptor polymorphisms as possible explanations10,11. In conclusion, polymyositis is a rare disease with several epidemiologic features and serologic markers which are neither sensitive nor specific, but key history and physical exam findings can help arrive at a diagnosis of polymyositis when confronted with a case of myasthenia and an elevated CPK, and thus aid in promptly initiating appropriate therapy.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details was obtained from the patient.", "appendix": "Author contributions\n\n\n\nSA conceived the case report. HK prepared the first draft of the case report. All authors were involved in the revision of the drafts 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\nHuerta-Alardin AL, Varon J, Marik PE: Bench-to-bedside review: Rhabdomyolysis -- an overview for clinicians. Crit Care. 2005; 9(2): 158–169. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nErnste FC, Reed AM: Idiopathic inflammatory myopathies: current trends in pathogenesis, clinical features, and up-to-date treatment recommendations. Mayo Clin Proc. 2013; 88(1): 83–105. PubMed Abstract \| Publisher Full Text\n\nRayavarapu S, Coley W, Kinder TB, et al.: Idiopathic inflammatory myopathies: pathogenic mechanisms of muscle weakness. Skelet Muscle. 2013; 3(1): 13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBriemberg HR, Amato AA: Dermatomyositis and polymyositis. Curr Treat Options Neurol. 2003; 5(5): 349–356. PubMed Abstract \| Publisher Full Text\n\nDalakas MC, Hohlfeld R: Polymyositis and dermatomyositis. Lancet. 2003; 362(9388): 971–982. PubMed Abstract \| Publisher Full Text\n\nFindlay AR, Goyal NA, Mozaffar T: An overview of polymyositis and dermatomyositis. Muscle Nerve. 2015; 51(5): 638–656. PubMed Abstract \| Publisher Full Text\n\nMammen AL: Dermatomyositis and polymyositis: Clinical presentation, autoantibodies, and pathogenesis. Ann N Y Acad Sci. 2010; 1184: 134–153. PubMed Abstract \| Publisher Full Text\n\nBohan A, Peter JB: Polymyositis and dermatomyositis (first of two parts). N Engl J Med. 1975; 292(7): 344–347. PubMed Abstract \| Publisher Full Text\n\nMohan C, Assassi S: Biomarkers in rheumatic diseases: how can they facilitate diagnosis and assessment of disease activity? BMJ. 2015; 351: h5079. PubMed Abstract \| Publisher Full Text\n\nBonifati DM, Witchel SF, Ermani M, et al.: The glucocorticoid receptor N363S polymorphism and steroid response in Duchenne dystrophy. J Neurol Neurosurg Psychiatry. 2006; 77(10): 1177–1179. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIkezoe K, Ohshima S, Osoegawa M, et al.: Expression of granulysin in polymyositis and inclusion-body myositis. J Neurol Neurosurg Psychiatry. 2006; 77(10): 1187–1190. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "13251", "date": "05 May 2016", "name": "Martin Aringer", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 educational case of a 64 year old male who presented with a fairly typical picture of a few weeks of proximal muscle weakness, difficulties swallowing, increased AST and very high CPK levels, but was initially misdiagnosed as having rhabdomyolysis. The absence of a usual trigger of rhabdomyolysis, the lack of rapid improvement and the pharyngeal involvement were early clues that the latter differential diagnosis was not correct. A muscle biopsy was unfortunately negative, and the authors found no specific autoantibodies, so that the diagnosis is not certain. The clinical picture, the MRI findings, and the improvement under glucocorticoids, which usually takes at least two weeks, nevertheless support that this patient has polymyositis. The only unusual finding is the normal ESR. In addition, the authors should have tested for antinuclear antibodies (ANA) by indirect immunofluorescence on HEp2 cells, where both ANA and cytoplasmic fluorescence would have given further clues. Many of the patients with anti-synthetase antibodies do have additional symptoms, ranging from mild Raynaud’s and mild arthritis to mechanics’ hand and early signs of interstitial lung disease. In addition to anti-Jo-1 antibodies, several other anti-synthetase antibodies can be tested for today. Some patients will have malignancies underlying new onset polymyositis or, more often dermatomyositis.Recommendations for (small) changes:understood (instead of understand) in the last sentence of the Introduction.in the setting of very high CPK values… (instead of elevated CPK values) second sentence of the Discussion.The final criterion (instead of criteria) in the middle of the first paragraph of the Discussion; a reference should also be added there.Raynaud’s (instead of Raynaud) and systemic sclerosis (instead of scleroderma or CREST) in the second paragraph of the Discussion", "responses": [] }, { "id": "15273", "date": "28 Jul 2016", "name": "Xenofon Baraliakos", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 of a differential diagnosis for inflammatory myopathy. I only have minor suggestions or open questions.\nWas ESR only tested once? What about previous examinations which might have shown increased values? In daily practice we ask for such results when we see these patients (e.g. from previous GP consultations) and it would be interesting to see if the patient never has had increased inflammatory markers, which would be a bit unusual.\n\nShould there be an MR image of the pathologic finding, it would be nice to add it to this report, if the space allows.\n\nDid you ever consider doing a second biopsy for the case that the right area has been missed? If not, it would be worth it to add a sentence in the Discussion about the role of repeated biopsies in such cases.\n\nThe patient had reported to have poorly controlled diabetes mellitus. What happened to that condition after 1 month of high-doses of cortisone and how did you deal with it? Again, 1-2 sentences in the Discussion would be informative in order to think of how to solve this comorbidity.", "responses": [] } ]	1	https://f1000research.com/articles/5-164
https://f1000research.com/articles/5-163/v1	11 Feb 16	{ "type": "Review", "title": "The business of deubiquitination – location, location, location", "authors": [ "Erin S. Coyne", "Simon S. Wing", "Erin S. Coyne" ], "abstract": "A majority of proteins in the cell can be modified by ubiquitination, thereby altering their function or stability. This ubiquitination is controlled by both ubiquitinating and deubiquitinating enzymes (DUBs). The number of ubiquitin ligases exceeds that of DUBs by about eightfold, indicating that DUBs may have much broader substrate specificity. Despite this, DUBs have been shown to have quite specific physiological functions. This functional specificity is likely due to very precise regulation of activity arising from the sophisticated use of all mechanisms of enzyme regulation. In this commentary, we briefly review key features of DUBs with more emphasis on regulation. In particular, we focus on localization of the enzymes as a critical regulatory mechanism which when integrated with control of expression, substrate activation, allosteric regulation, and post-translational modifications results in precise spatial and temporal deubiquitination of proteins and therefore specific physiological functions. Identification of compounds that target the structural elements in DUBs that dictate localization may be a more promising approach to development of drugs with specificity of action than targeting the enzymatic activity, which for most DUBs is dependent on a thiol group that can react non-specifically with many compounds in large-scale screening.", "keywords": [ "deubiquitination", "deubiquitinating enzymes", "DUB" ], "content": "Introduction\n\nThe conjugation of the 76 amino acid peptide ubiquitin to proteins is an important post-translational modification that can modulate most if not all cellular processes. This occurs via the consecutive action of three enzymes: E1 ubiquitin-activating enzymes (two mammalian genes), E2 ubiquitin conjugating enzymes (~35 genes), and E3 ubiquitin ligases (~750 genes)1. The E3 ligases are critical for conferring substrate specificity (reviewed in 2). Ubiquitin is covalently linked by its C-terminal glycine to, most commonly, the ε-amino group of a lysine on a target protein through an isopeptide bond. Occasionally, ubiquitin can be conjugated to cysteine, serine, threonine, and N-terminal methionine residues3–7. Subsequent ubiquitin moieties can be covalently linked to one of the seven lysine residues or the N-terminal methionine on the proximal ubiquitin to generate a polyubiquitin chain. Distinct functions are conferred depending on whether the protein is monoubiquitinated or polyubiquitinated and on the type of chain linkage. The two most common chain linkage types, K48 and K63, typically direct the substrate to different outcomes; the former is usually targeting proteins for degradation by the 26S proteasome, whereas the latter is generally involved in signal transduction, DNA repair, or endosomal sorting8–10. Conjugation of a linear chain of ubiquitins linked via their N-terminal methionines can serve to recruit proteins in cytokine signaling11,12. K11-linked chains13 and probably the remaining chain types14 can also target proteins to the proteasome. The 26S proteasome is a key mediator of intracellular protein homeostasis. It is an approximately 2.5-MDa macromolecular complex comprising a 20S cylindrical core particle capped at both ends by 19S regulatory particles. The 19S cap serves to recognize ubiquitinated substrates and allow their translocation into the lumen of the 20S core particle where the substrate then is hydrolyzed by the proteolytic machinery (reviewed in 2).\n\nImportantly, ubiquitination is dynamic and reversible. Whereas early studies focused on understanding how ubiquitin is conjugated to substrates, recent years have seen markedly increased interest in deubiquitinating enzymes (DUBs). These studies have demonstrated many functions for deubiquitination, giving support to the notion that DUBs play equally important roles as ligases do in controlling ubiquitination.\n\nA number of excellent reviews on DUBs have been published recently15–18. Therefore, in this commentary, we will highlight only the key concepts regarding the structure, functions, and mechanisms of these enzymes as these have been discussed in detail elsewhere. Instead, we will focus on the regulation of DUBs with emphasis on the role of localization in complexes and subcellular organelles in modulating their activities and function. We believe that such localization is a central factor in explaining how a relatively limited number of DUBs can exert a large range of specific functions. We will also comment briefly on the implications of this information on strategies for targeting DUBs for therapeutic purposes.\n\n\nStructure and mechanism\n\nDUBs are peptidases that catalyze the cleavage of the bond formed between ubiquitin and substrate or ubiquitin and ubiquitin. There are approximately 90 DUBs in the human genome, compared with the more than 750 E3 ligases1. There are five DUB families classified on the basis of the homology of their catalytic domains. These families are the ubiquitin C-terminal hydrolase (UCH), ubiquitin-specific protease (USP), ovarian tumor domain (OTU), Machado-Joseph disease (MJD), and Jab1/Mpn/Mov34 (JAMM) enzymes (Table 1). All of these families are cysteine proteases, except for JAMM family members, which are metalloproteases. The crystal structures of members of each of these families have been solved (reviewed in 19). The catalytic mechanism of the cysteine protease DUBs is similar to that of plant papains, whereby cysteine, histidine, and aspartate residues form a catalytic triad where the histidine primes the cysteine for nucleophilic attack on the peptide bond and the aspartate aligns and polarizes the histidine. Metalloproteases require the co-ordination of a zinc ion for catalysis, which allows the abstraction of a hydrogen atom from a water molecule, generating a reactive hydroxyl ion capable of attacking the peptide bond.\n\nEnzymes cited in this commentary are shown in bold.\n\n\nFunction\n\nMaintaining an adequate pool of free ubiquitin available for immediate conjugation is essential for the ability of the ubiquitin system to respond rapidly to changing cellular needs. DUBs play several critical roles in this general function of maintaining free ubiquitin. Ubiquitin is encoded in the human genome as four distinct genes: the two polyubiquitin genes UBB and UBC and the ubiquitin-fusion genes UBA52 and RPS27A, which encode a single ubiquitin protein fused to the ribosomal proteins L40 and S27A, respectively. Thus, ubiquitin is synthesized de novo as fusion proteins that must be cleaved to generate free ubiquitin by DUBs. The free ubiquitin pool is also maintained by recycling ubiquitin that has been released from proteins just prior to destruction by either the proteasome or the endocytic, lysosomal pathway. DUBs act at both locations to provide this recycling function (see below). Finally, DUBs also contribute ubiquitin to the free pool through their removal of ubiquitin from specific substrates with the effect of reversing or preventing the effects of ubiquitination. DUBs can also act to edit or remodel ubiquitin chains on substrates20 and thus may redirect their fate21,22. The extent to which DUBs play such a remodeling role remains unknown. But the identification of DUBs which act on specific chain linkages—e.g. AMSH23 and Ataxin320 for Lys 63-linked ubiquitin—clearly makes such a function plausible. Interestingly, DUBs can also inhibit conjugation by binding to the E2 and interfering with ubiquitin transfer to the E324,25.\n\n\nRegulation\n\nThe many fewer DUBs compared with ubiquitin ligases suggest that DUBs may have much broader specificity with many more substrates per DUB than per ligase. Therefore, regulation of their activity is critical to maintain specificity and occurs through both intramolecular and external factors (reviewed in Sahtoe and Sixma18). Indeed, evidence to date indicates that DUBs employ all the classic mechanisms of enzyme regulation in sophisticated fashions.\n\nRegulation of expression is well documented. Variation of expression of some enzymes in tissue(s)/cell type15 or upon specific stimuli26 represents one layer of control which can allow DUBs to have specific effects. Such regulation of expression takes place through both transcriptional and post-transcriptional mechanisms, including regulation by miRNAs27. Furthermore, regulation of enzyme levels by cleavage or degradation also occurs. USP1 cleaves itself following ultraviolet irradiation, leading to accumulation of the DNA replication processivity factor PCNA28. The OTU domain containing A20 can be inactivated by cleavage by MALT1, a protein associated with mucosa-associated lymphoid tissue lymphoma29. A number of DUBs exist in complex with E3s. The DUBs often can modulate ubiquitination of the E330–32 as well as ubiquitination by the E3—whether of itself or other substrates (e.g. USP7 on p5333 and its ligase Mdm234)—but the E3 can also modulate the stability of the DUB35.\n\nSubstrate activation – The apo enzymes are often in an inactive state and this is due to being in a conformation that does not allow catalysis or due to auto-inhibitory loops that impair substrate access to the active site. Binding of the substrate36 or the ubiquitin portion of the substrate37 can result in reorganization of the enzyme into a conformationally active form, indicating that substrate activation is an important regulatory mechanism.\n\nRegulation by post-translational modification is also well described with examples of modulation of activity by phosphorylation38,39, sumoylation40, and ubiquitination41,42. Furthermore, recent studies indicate that reactive oxygen species can inactivate many DUBs in a reversible manner by oxidizing the active-site cysteine to a cysteine sulphenic acid or sulphene amide43–45.\n\nAllosteric regulation due to binding of other proteins to the DUB is well described with examples of both activation46,47 and inhibition48,49 of enzyme activity.\n\nLocalization of the enzyme is becoming an increasingly appreciated regulatory mechanism allowing action on substrates that might not otherwise occur at significant rates if both enzyme and substrate were freely circulating, diluted in the cytoplasm. Here, we will highlight a few well-developed examples of localization – either to intracellular complexes or to organelles (Table 2) as a regulatory layer for DUB function. In many cases, the localization to a complex also results in allosteric regulation of the enzyme. A systematic study of localization of GFP-tagged DUBs indicates that approximately 25% of the enzymes are found in specific subcellular structures50.\n\nJAMM, JAB1/MPN/Mov34; MJD, Machado-Joseph disease; UCH, ubiquitin C-terminal hydrolase; USP, ubiquitin-specific protease.\n\nThe DUBs PMSD14/Rpn11, UCHL5/UCH37, and USP14 are all associated with the 19S regulatory cap of the proteasome; PMSD14 is a constituent component, whereas UCHL5 and USP14 are reversibly associated proteins. Upon binding to the 19S cap, UCHL5 and USP14 undergo restructuring, resulting in greatly increased enzymatic activity. Binding of UCHL5 to the proteasome repositions a crossover loop, thereby relieving an auto-inhibitory effect and allowing substrate access to the active site48. Similarly, in USP14, the ubiquitin-binding pocket is obscured by two loops and binding to the 19S regulatory cap reveals the ubiquitin-binding site necessary for deubiquitination51. The localization of these DUBs gives them specific access to substrates associated with the proteasome. PMSD14 cleaves the ubiquitin chain at its junction with the substrate, thereby allowing efficient unwinding and insertion of the substrate into the 20S core and so its DUB activity promotes proteolysis. Both USP14 and UCHL5 have been shown to deubiquitinate and impair proteolysis of some substrates. However, it remains possible that, for some substrates, these enzymes may edit chains into forms which allow more effective binding to or processing by the proteasome.\n\nReceptor endocytosis followed by either recycling to the plasma membrane or trafficking through multi-vesicular bodies (MVBs) to lysosomes for degradation plays an important role in modulating signal transduction. During endocytosis, monoubiquitination and polyubiquitination of the receptor constitute a sorting signal that can be decoded by the ESCRT (endosomal sorting complex required for transport) complexes. The four ESCRT complexes (ESCRT-0, -I, -II, and -III) function together to generate MVBs by allowing the remodeling of the plasma membrane and the budding and internalization of cargo-bearing vesicles. The contents of MVBs then are sent to the lysosome for degradation. Two DUBs are known to associate with ESCRT complexes in the endocytic/lysosomal pathway. AMSH (a JAMM family member with specificity for K63 chains) and USP8 associate with STAM proteins, a component of the ESCRT-0 complex52,53. AMSH and USP8 can also interact with CHMP proteins that are components of the late ESCRT-III machinery23,54. Association with ESCRT is necessary for AMSH function in endocytosis55 and disruption of this association causes accumulation of the EGF receptor via impaired degradation56. Thus, AMSH activity enhances receptor trafficking toward lysosomal degradation. USP8 can be both a positive and a negative regulator of receptor endocytosis. Loss of USP8 leads to hyperubiquitination and enhanced degradation of EGF, MET, and ERBB3 receptors57,58 but increases the level of the Wnt receptor Frizzled by enhancing receptor recycling59. The contrasting effects of loss of AMSH and USP8 on receptor stability as well as their differences in chain linkage specificity indicate that these ESCRT-associated DUBs have distinct functions.\n\nMitochondrial dysfunction can have profound effects on cell function and viability. Indeed, mitochondrial dysfunction and impaired clearance of damaged mitochondria are hallmarks of the neurodegenerative disorder Parkinson’s disease. The ubiquitin ligase Parkin is mutated in an autosomal recessive form of the disease. In Parkin-mediated mitochondrial clearance (mitophagy), the kinase PINK1 accumulates on damaged mitochondria and recruits Parkin to ubiquitinate a variety of substrates on the mitochondria (reviewed in 60). USP30 was first identified as a DUB with a mitochondrial targeting sequence that is embedded in the mitochondrial outer membrane and plays a role in mitochondrial dynamics61. Subsequent studies revealed that it is a negative regulator of mitophagy. USP30 antagonizes Parkin-mediated mitophagy by deubiquitinating its target substrates62. It has also been shown to delay the recruitment of Parkin to damaged mitochondria63. Other DUBs may also co-localize to the mitochondria and play a role in mitophagy. USP15 antagonizes Parkin mitochondrial ubiquitination64, USP35 can delay Parkin-mediated mitophagy through unclear mechanisms63, and USP8 removes K6-linked polyubiquitin chains from Parkin itself to facilitate its recruitment to damaged mitochondria65. Additionally, ubiquitin itself can be phosphorylated on serine 65 by PINK166–68. Interestingly, mitochondrial DUBs, including USP30, USP8, and USP15, are impaired at hydrolyzing these phosphoUb chains69, thus providing an additional regulatory mechanism for Parkin-mediated mitophagy.\n\nUSP19 was first identified as a DUB upregulated in skeletal muscle during catabolic conditions70. It is expressed as two major isoforms: one cytoplasmic and the other with a transmembrane domain that results in anchoring of the C-terminal tail of the protein in the endoplasmic reticulum (ER) membrane with retention of the catalytic domain in the cytoplasm71. Overexpression of USP19 has been shown to rescue model substrates from ER-associated degradation (ERAD)71 as well as an ER-localized ligase MARCH672. However, silencing of USP19 does not affect levels of ERAD substrates in a consistent manner72,73, so its physiological functions at the ER remain unclear. USP19 can inhibit myogenic differentiation through suppression of an unfolded protein response that is required for muscle cell fusion74. Interestingly, these effects are dependent on catalytic activity and occur with the ER but not the cytoplasmic isoform although both isoforms’ catalytic domains are in the cytoplasm, indicating that the localization is critical for its ability to deubiquitinate specific substrates74.\n\nHistone modifications are critical for DNA-dependent processes, including repair, replication, and transcription. Many DUBs have been shown to remove ubiquitin from chromatin, most commonly from histones H2A and H2B. MYSM1 and BRCC36 are two JAMM family members known to deubiquitinate H2A, with BRCC36 preferentially removing K63 polyubiquitin and suppressing DNA repair pathways. Importantly, MYSM1 and BRCC36 are associated with complexes that activate their deubiquitinating activity. MYSM1 is active as part of the 2A-DUB complex75, whereas BRCC36 is associated with the BRCA1-A complex and this association activates its activity in the nucleus76. Other DUBs capable of deubiquitinating histones include USP3, USP7, USP12, USP16, USP21, USP22, and USP44. Disruption of some of these DUBs results in altered cell cycle progression. Although USP3 and USP16 are not known to associate with any complexes, depletion of either enzyme results in aberrant cell cycle progression, with USP16 knockdown resulting in impaired mitosis77 and depletion of USP3 resulting in a delay in S-phase progression78. These different outcomes suggest that localization of DUBs to specific chromatin loci results in differential gene expression. DUBs could also target non-histone substrates at these sites to contribute to the phenotype.\n\n\nPharmacological targeting\n\nGiven the role of ubiquitination in many important processes that are deranged in disease, it is an attractive set of enzymes for pharmacological intervention. Targeting deubiquitination is also alluring in that the more limited number of DUBs in comparison with ligases makes phenotypic screens of DUBs more feasible. Indeed, the availability of small hairpin RNA (shRNA) or small interfering RNA (siRNA) libraries targeting all of the DUBs allows relatively rapid determination of whether loss of function of a DUB will yield a desirable phenotype. The limited number of DUBs allows screening to be applied to even relatively low-throughput assays.\n\nOnce loss of function of a DUB is shown to produce an effect that might be clinically desirable, the development of that observation into a potential drug is much more challenging. Although a number of assays of DUB activity are available and amenable to high-throughput screens of large compound libraries and have resulted in some lead compounds (reviewed in 79), these assays are at high risk of yielding many unproductive hits as almost all of the DUBs are thiol-based proteases and the highly reactive thiol group of the catalytic cysteine residue is well recognized to react non-specifically to many compounds. Inhibition of the enzymatic activity may not be desirable for other reasons. The large number of potential substrates for each DUB may lead to many more undesirable effects arising from inhibition of catalysis. Whole body gene inactivation of the enzyme in mice may be helpful in predicting the extent of such adverse effects.\n\nA number of alternative strategies can be proposed to inhibit DUBs; however, these strategies require significantly more characterization of the enzymes. Since the enzymes must bind ubiquitin, an alternative approach to broad inhibition of a particular enzyme would be to inhibit its ability to bind ubiquitin. Generally, such inhibition would require knowledge of the structure of the enzyme bound to ubiquitin to identify the essential elements of the ubiquitin-binding domain(s). A recent mutagenic strategy has created ubiquitin variants that are able to bind and inhibit enzymatic activity80. Remarkably, variants that were selective for specific DUBs were obtained. Crystallization of the ubiquitin variant/DUB complex identified specific residues on the DUB that contact the variant and that yield specificity for a particular enzyme. Some DUBs have multiple ubiquitin-binding sites that permit binding of ubiquitin chains. These likely restrict the orientation of the ubiquitins in the chain that can be accommodated and therefore result in specificity of the enzyme for particular chain cleavages. Thus, inhibition of one of these specific domains may result in interference with ubiquitin chain binding or with the chain linkage specificity of the enzyme.\n\nTargeting specific functions of a DUB may be achieved by identification of the specific substrates that mediate these effects. Subsequent structure function analyses then can identify the interacting domains of the enzyme and substrate and lead to the development of assays that can screen for compounds that interfere with the interaction. As discussed earlier, an important determining factor for both substrate specificity and regulation is the localization of the DUB. Once the mechanisms that result in targeting to a specific compartment or complex are determined, then assays that measure this binding can be similarly designed to screen for inhibitors. Since there are several examples in which complex formation or substrate binding activates the DUB, inhibitors that stabilize the enzyme in the auto-inhibited form could be developed. All of these approaches will rely heavily on structural studies both to help design the assay and to confirm that the identified compounds are functioning through the expected mechanisms.\n\n\nClosing perspectives\n\nMuch progress has been made in our understanding of DUBs over the past 15 years. Structures have now been solved for many DUBs. However, in a number of cases, only the catalytic domain has been resolved and it is clear that important information resides in the other regions of the enzyme and will be needed to improve success in pharmacological targeting. A much larger gap in our understanding is in the functions of the enzymes, both at a molecular level (substrate identification) and at a cellular or whole organismal level (the physiological effects of removal of ubiquitin from the substrates). The former may be addressed through analysis of differential ubiquitination of proteins upon loss of function of the DUB. Although much progress has been made in ubiquitinome analysis, it remains to be determined whether current methods are sufficiently reproducible and precise to detect what might be small differences in steady-state levels of ubiquitination that arise upon DUB inactivation. The availability and application of methods for RNA silencing, gene editing, and gene knockout have been transformative in permitting the elucidation of physiological functions. However, such information remains available for only a small minority of the DUBs. Finally, our understanding of regulation of DUBs has progressed significantly. All of the classic types of enzyme regulation are present, but localization within organelles and complexes, allosteric regulation within complexes, and substrate activation appear to be prominent mechanisms which together allow tightly regulated activity on specific substrates. Identifying the structural elements underlying these mechanisms will offer the potential of targeting them to obtain drugs with highly specific effects.", "appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nWork in the authors' laboratory cited here was supported by grants from the Canadian Institutes of Health Research.\n\n\nReferences\n\nHutchins AP, Liu S, Diez D, et al.: The repertoires of ubiquitinating and deubiquitinating enzymes in eukaryotic genomes. Mol Biol Evol. 2013; 30(5): 1172–87. 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PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nNijman SM, Huang TT, Dirac AM, et al.: The deubiquitinating enzyme USP1 regulates the Fanconi anemia pathway. Mol Cell. 2005; 17(3): 331–9. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nSong EJ, Werner SL, Neubauer J, et al.: The Prp19 complex and the Usp4Sart3 deubiquitinating enzyme control reversible ubiquitination at the spliceosome. Genes Dev. 2010; 24(13): 1434–47. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvan Loosdregt J, Fleskens V, Fu J, et al.: Stabilization of the transcription factor Foxp3 by the deubiquitinase USP7 increases Treg-cell-suppressive capacity. Immunity. 2013; 39(2): 259–71. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nGarcía-Santisteban I, Bañuelos S, Rodríguez JA: A global survey of CRM1-dependent nuclear export sequences in the human deubiquitinase family. Biochem J. 2012; 441(1): 209–17. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nXiong J, Wang Y, Gong Z, et al.: Identification of a functional nuclear localization signal within the human USP22 protein. Biochem Biophys Res Commun. 2014; 449(1): 14–8. PubMed Abstract \| Publisher Full Text\n\nDirac AM, Bernards R: The deubiquitinating enzyme USP26 is a regulator of androgen receptor signaling. Mol Cancer Res. 2010; 8(6): 844–54. PubMed Abstract \| Publisher Full Text\n\nPopov N, Wanzel M, Madiredjo M, et al.: The ubiquitin-specific protease USP28 is required for MYC stability. Nat Cell Biol. 2007; 9(7): 765–74. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nSun XX, He X, Yin L, et al.: The nucleolar ubiquitin-specific protease USP36 deubiquitinates and stabilizes c-Myc. Proc Natl Acad Sci U S A. 2015; 112(12): 3734–9. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nMakarova OV, Makarov EM, Lührmann R: The 65 and 110 kDa SR-related proteins of the U4/U6.U5 tri-snRNP are essential for the assembly of mature spliceosomes. EMBO J. 2001; 20(10): 2553–63. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSuresh B, Ramakrishna S, Lee HJ, et al.: K48- and K63-linked polyubiquitination of deubiquitinating enzyme USP44. Cell Biol Int. 2010; 34(8): 799–808. PubMed Abstract \| Publisher Full Text\n\nYao T, Song L, Jin J, et al.: Distinct modes of regulation of the Uch37 deubiquitinating enzyme in the proteasome and in the Ino80 chromatin-remodeling complex. Mol Cell. 2008; 31(6): 909–17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMashtalir N, Daou S, Barbour H, et al.: Autodeubiquitination protects the tumor suppressor BAP1 from cytoplasmic sequestration mediated by the atypical ubiquitin ligase UBE2O. Mol Cell. 2014; 54(3): 392–406. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nTait D, Riccio M, Sittler A, et al.: Ataxin-3 is transported into the nucleus and associates with the nuclear matrix. Hum Mol Genet. 1998; 7(6): 991–7. PubMed Abstract \| Publisher Full Text\n\nCurcio-Morelli C, Zavacki AM, Christofollete M, et al.: Deubiquitination of type 2 iodothyronine deiodinase by von Hippel-Lindau protein-interacting deubiquitinating enzymes regulates thyroid hormone activation. J Clin Invest. 2003; 112(2): 189–96. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nBlount JR, Burr AA, Denuc A, et al.: Ubiquitin-specific protease 25 functions in Endoplasmic Reticulum-associated degradation. PLoS One. 2012; 7(5): e36542. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nLiu Z, Meray RK, Grammatopoulos TN, et al.: Membrane-associated farnesylated UCH-L1 promotes alpha-synuclein neurotoxicity and is a therapeutic target for Parkinson's disease. Proc Natl Acad Sci U S A. 2009; 106(12): 4635–40. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAkhavantabasi S, Akman HB, Sapmaz A, et al.: USP32 is an active, membrane-bound ubiquitin protease overexpressed in breast cancers. Mamm Genome. 2010; 21(7–8): 388–97. PubMed Abstract \| Publisher Full Text\n\nThorne C, Eccles RL, Coulson JM, et al.: Isoform-specific localization of the deubiquitinase USP33 to the Golgi apparatus. Traffic. 2011; 12(11): 1563–74. PubMed Abstract \| Publisher Full Text" }	[ { "id": "12400", "date": "11 Feb 2016", "name": "Henry L. Paulson", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12401", "date": "11 Feb 2016", "name": "Kazuhiro Iwai", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-163
https://f1000research.com/articles/5-162/v1	11 Feb 16	{ "type": "Review", "title": "Recent Advances in the Trichomonas vaginalis Field", "authors": [ "David Leitsch" ], "abstract": "The microaerophilic protist parasite Trichomonas vaginalis is occurring globally and causes infections in the urogenital tract in humans, a condition termed trichomoniasis. In fact, trichomoniasis is the most prevalent non-viral sexually transmitted disease with more than 250 million people infected every year. Although trichomoniasis is not life threatening in itself, it can be debilitating and increases the risk of adverse pregnancy outcomes, HIV infection, and, possibly, neoplasias in the prostate and the cervix. Apart from its role as a pathogen, T. vaginalis is also a fascinating organism with a surprisingly large genome for a parasite, i.e. larger than 160 Mb, and a physiology adapted to its microaerophilic lifestyle. In particular, the hydrogenosome, a mitochondria-derived organelle that produces hydrogen, has attracted much interest in the last few decades and rendered T. vaginalis a model organism for eukaryotic evolution.This review will give a succinct overview of the major advances in the T. vaginalis field in the last few years.", "keywords": [ "Trichomonas vaginalis", "trichomoniasis", "non-viral sexually transmitted disease", "hydrogenosome", "metronidazole" ], "content": "Introduction\n\nTrichomonas vaginalis (Tv) is a globally occurring anaerobic/microaerophilic protist parasite which colonizes the epithelium of the human urogenital tract. Although often asymptomatic, Tv infections can cause inflammation in the cervix, the vagina, and the urethra. Based on estimates of the World Health Organization (WHO) from 20081, trichomoniasis constitutes the most prevalent non-viral sexually transmitted disease (STD) worldwide, affecting more than 276 million people every year. Women and men are infected with comparable frequency, but in men symptoms are normally mild and infections are cleared by the host’s immune system within weeks. In women, however, Tv infections can persist for many years, and symptoms, mainly pruritus caused by inflammation and odorous vaginal discharge, can attain a severity which is debilitating. As trichomoniasis is not a life-threatening disease, it was often belittled as a “nuisance infection” in the past. A large number of studies from the last 20 years or so, however, have shown that underlying Tv infections increase the risk of adverse pregnancy outcomes and contagion with HIV virus2. Given the fact that HIV and Tv are often jointly epidemic in many parts of the world, this is an alarming finding. Moreover, a higher risk of developing prostate cancer due to Tv infection has been suggested2.\n\nBefore the development of the 5-nitroimidazole drug metronidazole in 1960, trichomoniasis was notoriously difficult to treat. Nowadays, most patients can be successfully treated with metronidazole or another more effective 5-nitroimidazole derivative, tinidazole. However, resistance to 5-nitroimidazoles does occur and seems to be on the rise3. In addition, allergic reactions to nitroimidazoles have been reported and side effects of nitroimidazole treatment can be disturbing.\n\nApart from its role as a pathogen, Tv has attracted great interest from geneticists, biochemists, and evolutionary biologists after the discovery of the hydrogenosome4, a mitochondrion-like organelle which generates hydrogen. Due to its microaerophilic lifestyle, Tv does not have the ability to generate ATP by oxidative phosphorylation but depends on substrate-level phosphorylation. Originally, it was assumed that the hydrogenosome is an ancestral form of the mitochondrion5, which kindled interest in Tv as an assumed archaic eukaryote. This notion, however, has been thoroughly revised after publication of the Tv genome in 20076. It is now apparent, based on phylogenetic studies, that the hydrogenosome constitutes a reduced form of fully developed mitochondria. Nevertheless, from the evolutionary and cell biologist’s point of view, the hydrogenosome remains an intriguing organelle, and the extraordinary size of the Tv genome, exceeding 160 Mb, will certainly provoke further research in the years to come.\n\nIn this review, I will give a brief but comprehensive overview of the advances in the research on Tv from the last 5 years or so, spanning from the epidemiology to the infection biology, treatment, and cell biology of this fascinating parasite.\n\n\nEpidemiology\n\nAlthough Tv is a worldwide occurring parasite, prevalence rates differ very strongly in different parts of the world. In the Americas, for example, its incidence is calculated to be as high as 180 per 1000 men and women, whereas in South-East Asia estimates are much lower, with 40 to 50 per 1000 men and women7. In total, 276 million infections with Tv are believed to occur worldwide and per annum1. These numbers are very high indeed, but it is estimated that most Tv infections (up to 80%) are asymptomatic8. Importantly, men are infected equally frequently, but 89% of trichomoniasis cases are actually diagnosed in women because of their higher incidence of symptoms, which are sometimes severe and debilitating. The main concern about Tv infections, however, is their predisposing nature for other diseases or sequelae2, and a number of new studies give justification to this concern. For example, Tv was found to be associated with human papilloma virus infections and cervical cytological abnormalities9. Moreover, in a meta-analytical study, strong statistical evidence was presented for an association of an underlying Tv infection and preterm birth10. Most importantly, however, evidence for a predisposition for infection with HIV in Tv-infected individuals is mounting. In a meta-study on 31 studies, it was concluded that the risk of HIV acquisition is increased 2- to 3-fold in Tv carriers11, and it was found that Tv infection increased the risk of HIV infection 2.5-fold in macaques, which serve as a non-human primate model. Accordingly, it was calculated that annual screening for Tv would save US$553 per woman and lifetime in the prevention of new HIV infections to susceptible male partners in the United States alone12.\n\nIn order to get a picture of the genetic diversity of Tv, a large-scale study13 was conducted, subjecting 235 Tv isolates, collected from all around the globe, to microsatellite genotyping14. Intriguingly, Tv was found to group into two distinct clusters or “types”. Both types occur worldwide with comparable frequency, although type 1 is presumably the older clade13. Interestingly, the presence of Tv virus (TVV) is unequally distributed within the two types, with about 70% of all type 1 isolates, but only 30% of type 2 isolates, carrying the virus. Conversely, metronidazole resistance is far more prevalent in type 2 isolates.\n\n\nTreatment\n\nSince 1960, metronidazole and other 5-nitroimidazoles, such as tinidazole, have been the mainstay of Tv treatment3. 5-nitroimidazoles have been reported to damage DNA, form adducts with proteins (partly with inhibiting consequences15), and cause oxidative damage in the cell by depleting thiol pools15. 5-nitroimidazoles are in fact prodrugs, which have to be reduced at their nitro groups in order to become toxic. This reaction, however, takes place quantitatively only in microaerophilic/anaerobic organisms and has been suggested to be catalyzed by several enzymes and factors such as ferredoxin16, nitroreductase17, and thioredoxin reductase15. Resistance to 5-nitroimidazoles in clinical Tv isolates does occur, sometimes leading to extended and complex treatment regimens18. Clinical metronidazole resistance is based on decreased oxygen scavenging in the cell, leading to higher intracellular oxygen concentrations19. Accordingly, expression of flavin reductase 1, an enzyme that uses flavin mononucleotide (FMN) to reduce molecular oxygen to H2O2, has been described to be downregulated or even shut-off in metronidazole-resistant Tv20,21. In addition, a correlation between metronidazole resistance and mutations in the genes for nitroreductase 4 and 6 was found22.\n\nDue to the occurrence of Tv strains refractory to 5-nitroimidazole treatment, the search for alternative treatments has never stopped. In recent years, a number of promising alternatives were presented, including pentamycin23, boric acid24, N-chlorotaurine25, and drug-free chitosan26, all of which would have be to administered intravaginally and not systemically, as is the case with 5-nitroimidazoles. Further, a combination of metronidazole and miconazole, administered intravaginally, was shown to greatly reduce adverse side effects often reported for systemic metronidazole treatment27.\n\n\nPathogenicity\n\nThe last few years have seen a number of major advances in our understanding of Tv pathogenicity. In a number of studies, including proteomic and glycobiological approaches, several key components of the Tv cell surface were described. First, a detailed chemical structure of Tv lipoglycan, a surface molecule strongly binding to human galectin-1 and -328, was published29. Further, a large surface proteome study was performed30, identifying 261 putative membrane proteins, including ABC transporters and 11 BspA proteins. BspA proteins constitute a huge surface protein family in the Tv genome comprising 911 members31. They could bind to proteins of the extracellular matrix of the host epithelium, e.g. fibronectin, and elicit strong immune responses. In addition, this proteomic study revealed the existence of two hypothetical proteins which seem to enhance adhesion of Tv to the host epithelium30. Another proteomic study was performed using exosome-enriched cellular fractions of Tv, leading to the identification of 215 proteins, putatively localizing to exosome vesicles32. Among these proteins were one BspA-like protein and one tetraspanin. Tetraspanins are a protein family known to be involved in cell adhesion, and proteins that had before been suggested to be involved in adhesion of Tv to the epithelium, such as glyceraldehyde 3-phosphate dehydrogenase33, enolase34, succinyl-CoA synthetase35, and GP63 protease36. Importantly, a large-scale transcriptomic deep sequencing study (RNAseq) with Tv during early infection performed by another work group corroborated many of these observations37. Exosomes also contain large amounts of short RNA molecules (25–200 nucleotides) and enhance adhesion to vaginal ectocervical cells (VECs) when added extraneously to Tv strains with poor adhesion capacity32. It is important to note that cell adhesion is an absolutely necessary prerequisite for the lysis of host cells by Tv38. After cell adhesion has taken place, several factors are assumed to be involved in host cell lysis, including metalloproteases39,40, cysteine proteases41–43, a rhomboid protease (TvROM1)44, and phospholipase A245. Tv also secretes a migration inhibition factor (TvMIF)46 which can replace human migration factor (HuMIF) to trigger proinflammatory cytokine release. Possibly, this contributes to the increased risk of developing prostate cancer in Tv-infected men3.\n\nThe detection of tetraspanins in Tv exosomes prompted further research on this protein family47,48. Of the tetraspanins studied, all but one (TvTsp2) were strongly upregulated upon contact with VECs48. TvTsp6 changes its localization in the cell upon VEC contact and migrates from the flagellum to the plasma membrane. The C-terminal tail was found to be necessary for correct localization. Intriguingly, one tetraspanin, TvTSP8, seems to mediate Tv aggregation rather than VEC adhesion48. Contact with VECs also triggers a reorganization of the actin cytoskeleton and enables the rapid transition of flagellate to amoeboid morphology49. This process is mediated by TvFIM1, the only fimbrin found to be expressed in Tv.\n\nWhen discussing the pathogenicity of Tv, it is also important to take into account other microorganisms that coincide with the parasite, especially Mycoplasma hominis and TVV. In the presence of M. hominis, Tv infection triggers a far more pronounced proinflammatory reaction than in its absence50. The enhancing effect of TVV (which resides in about half of all Tv isolates) on the proinflammatory response seems to be even stronger51, as TVV is sensed by Toll-like receptor 3 on the surface of VECs. Especially worrying is the observation that metronidazole treatment, accompanied by the release of large amounts of virus particles from necrotic Tv, further amplifies this adverse response. The contents of this section are visualized in Figure 1.\n\nIn order to exert a cytopathic effect, it is necessary37 that Tv (light blue) binds (1) to the extracellular matrix (light green) or the host epithelium (dark green). Binding is accomplished by several surface proteins and other surface molecules that bind to a structure on the host’s cell surface. Among these are lipoglycan27, BspA28,30, tetraspanins28,45,46 and several others, such as glyceraldehyde 3-phosphate dehydrogenase32, enolase33, and succinyl-CoA synthetase34 on the Tv surface, galectins-1 and -328 on the host cell surface, and fibronectin32 in the extracellular matrix. Several Tv factors necessary for adhesion to the host epithelium reach the Tv surface or the epithelium surface via exosomes31 (2). Damage to the host cell is caused by several effectors (3), including cysteine proteases, metalloproteases, rhomboid proteases, and phospholipase A2. Tv migration inhibition factor might favor the development of neoplasia in the prostate44. In the presence of Mycoplasma hominis48 and Tv virus49 (4), symptoms might be exacerbated.\n\n\nBiochemistry and cell biology\n\nThe last few years have seen several transcriptomic and proteomic studies addressing the impact of growth and culture conditions on gene expression in Tv. Deep sequencing of RNA libraries was applied to identify genes that are differentially expressed under oxidative stress37 and glucose restriction52. Oxidative stress led to an upregulation of expression of 218 genes after 2 hours, including peroxiredoxins (Prx), thioredoxin reductase, thioredoxins, superoxide dismutases (SODs), rubrerythrin, and ferredoxins37. Upregulation of SOD and Prx upon oxidative stress at the protein level had already been reported before53, underpinning the validity of the transcriptomic approach. Interestingly, glucose starvation also led to upregulation of SOD, Prx, and rubrerythrin, resulting in a more H2O2-resistant phenotype52. Most glycolytic enzymes, however, were downregulated in glucose-starved cells, accompanied by a strong upregulation of glutamate dehydrogenase, which produces α-ketoglutarate by oxidative deamination of glutamate. Also, autophagy was observed in glucose-starved cells, and autophagy markers, i.e. autophagy-related genes (atg), were upregulated in expression52. In a phosphoproteomic study, 82 phosphoproteins were discovered in Tv54, a number conspicuously low given that more than 1000 genes for kinases exist in the Tv genome54,55.\n\nThe glycobiology of Tv has received considerable attention recently, and a comprehensive study on N-glycan composition in four Tv strains was published56. In all strains, a major core structure, a truncated oligomannose form (Man5GlcNAc2) with α1,2-mannose residues, could be identified. In contrast, modifications with phosphoethanolamine and terminal N-acetyllactosamine varied depending on the strain studied. Moreover, the core structure is often decorated with xylose29,56, which has been described as typical for trematodes and plants. Indeed, Tv encodes a functional UDP-xylose synthase57, the first to be described in a unicellular parasite. Further, asparagine-linked N-glycans of Tv were found to bind human mannose-binding lectin and retroviral lectins58.\n\nNaturally, the hydrogenosome, as a model for organelle evolution, has remained one of the major focuses in the Tv field. Again, proteomic studies provided a deeper insight into hydrogenosome biology. A study on hydrogenosomal membrane proteins, for example, demonstrated that hydrogenosomes and mitochondria have important core membrane components in common which are responsible for protein import and metabolite transport59. Hydrogenosomes also contain a dynamin-like protein which is likely to be involved in hydrogenosomal fission60. Nevertheless, essential differences with mitochondria also exist which can be attributed to the microaerophilic lifestyle and evolutionary adaptations of Tv and other related parasites. This is also reflected in the much lower number of proteins in the hydrogenosome61 as compared to mitochondria, i.e. about 500 vs. 1000–1500. The proteome’s composition is also rather variable, as the expression levels of many hydrogenosomal proteins were found to depend on available iron concentrations62,63. This is in line with the high abundance of iron-sulfur cluster proteins such as pyruvate:ferredoxin oxidoreductase, hydrogenase, and ferredoxin in this organelle. Unfortunately, it is hard to predict the localization of proteins to the hydrogenosome based on sequence information alone because protein import seems to depend on as yet poorly defined internal sequences, rather than on N-terminal targeting sequences. The latter seem, if at all existent, to be dispensable in most cases, likely due to the loss of the electrochemical gradient61,64,65. This difficulty can, however, be partly overcome by applying sophisticated machine learning approaches66. Also, other recent findings are difficult to put into perspective, e.g. the obvious functional redundancy of one of the most abundant proteins in the hydrogenosomal membrane, Tvhmp2367, or the localization of arginine deiminase to the hydrogenosome while other key enzymes of the arginine dihydrolase pathway reside in the cytosol68.\n\n\nGenomics and gene expression\n\nThe Tv genome is extremely large for a protist and might be even larger than originally anticipated, i.e. 175 Mb in size69 rather than 160 Mb6. Intriguingly, as much as 65% of its content consists of repetitive sequences, including transposable elements such as representatives of the types Maverick and Tc1/mariner70, and microRNA71. The expansion of gene families is a common phenomenon in Tv, so that the vast number of 60,000 genes has accumulated in the genome6. On the other hand, the proportion of pseudogenes seems to be extraordinarily large as well, with, for example, as much as 46% of the 123 transmembrane adenylyl cyclases being truncated or having nonsense mutations72. However, many pseudogenes are being transcribed, leading to a high representation of pseudogene mRNA in the long non-coding RNA pool73. In total, only about half of the annotated genes are being expressed but almost all gene families are represented73. It is likely that Tv harnesses this fluctuant nature of its genome to adaptive innovation, i.e. evolution. This flexibility might apply to annotated, functional genes as well. For example, seven full-length isoforms of the enzyme flavin reductase (FR1-7) with varying relatedness to each other are present in the genome21, but only FR1 has a Km for FMN which is low enough to be of plausible physiological importance. Three other FRs have high Vmax but also high Km, and the remaining three have low Vmax and high Km. Nevertheless, all of the less specific isoforms are expressed, if not in all strains, and can, at very high expression levels, partly substitute for FR1.\n\nThe last few years have also seen several advances in our understanding of gene expression in Tv. Especially well studied are the Myb-like transcription factors tvMyb1-3, which are known to bind to the promoter sites MRE-1/MRE-2r and MRE-2f of the hydrogenosomal malate dehydrogenase gene, also known as ap65-174. In the case of tvMyb3, the DNA-binding site was crystallized and its structure determined75. In a suite of excellent studies, the same group also revealed the mechanism of nuclear import of all three tvMybs76–78. Further, core promoter elements in Tv79 and polyadenylation signals80 were described. Finally, Tv mRNA was found to possess a metazoan/plant-like cap structure and a metazoan/plant-like capping enzyme81.\n\n\nConclusion\n\nIn recent years, considerable progress was achieved in the Tv field. Although there are still many open questions regarding Tv’s epidemiology, particularly in the context of facilitated HIV contagion and cancer, our understanding of Tv’s pathogenesis made a large leap forward and the picture is becoming ever more complete. In the treatment of Tv, several interesting alternatives, especially topical treatments, might eventually replace metronidazole, which potentially has worrying side effects. Finally, the genome of Tv has remained a fascinating colossus, whose complexity will trigger plenty of further research in the years to come.", "appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThis work was supported by project J3492 of the Austrian science fund (FWF).\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nWHO: Global incidence and prevalence of selected curable sexually transmitted infections – 2008. 2016. Reference Source\n\nKissinger P: Trichomonas vaginalis: a review of epidemiologic, clinical and treatment issues. BMC Infect Dis. 2015; 15: 307. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nSobel R, Sobel JD: Metronidazole for the treatment of vaginal infections. Expert Opin Pharmacother. 2015; 16(7): 1109–1115. 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PubMed Abstract \| Publisher Full Text\n\nLeitsch D, Drinić M, Kolarich D, et al.: Down-regulation of flavin reductase and alcohol dehydrogenase-1 (ADH1) in metronidazole-resistant isolates of Trichomonas vaginalis. Mol Biochem Parasitol. 2012; 183(2): 177–183. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLeitsch D, Janssen BD, Kolarich D, et al.: Trichomonas vaginalis flavin reductase 1 and its role in metronidazole resistance. Mol Microbiol. 2014; 91(1): 198–208. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPaulish-Miller TE, Augostini P, Schuyler JA, et al.: Trichomonas vaginalis metronidazole resistance is associated with single nucleotide polymorphisms in the nitroreductase genes ntr4Tv and ntr6Tv. Antimicrob Agents Chemother. 2014; 58(5): 2938–2943. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nKranzler M, Syrowatka M, Leitsch D, et al.: Pentamycin shows high efficacy against Trichomonas vaginalis. Int J Antimicrob Agents. 2015; 45(4): 434–437. PubMed Abstract \| Publisher Full Text\n\nBrittingham A, Wilson WA: The antimicrobial effect of boric acid on Trichomonas vaginalis. Sex Transm Dis. 2014; 41(12): 718–722. PubMed Abstract \| Publisher Full Text\n\nFürnkranz U, Nagl M, Gottardi W, et al.: In vitro activity of N-chlorotaurine (NCT) in combination with NH4Cl against Trichomonas vaginalis. Int J Antimicrob Agents. 2011; 37(2): 171–173. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPradines B, Bories C, Vauthier C, et al.: Drug-free chitosan coated poly(isobutylcyanoacrylate) nanoparticles are active against Trichomonas vaginalis and non-toxic towards pig vaginal mucosa. Pharm Res. 2015; 32(4): 1229–1236. PubMed Abstract \| Publisher Full Text\n\nSchwebke JR, Lensing SY, Sobel J: Intravaginal metronidazole/miconazole for the treatment of vaginal trichomoniasis. Sex Transm Dis. 2013; 40(9): 710–714. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nFichorova RN, Yamamoto HS, Fashemi T, et al.: Trichomonas vaginalis Lipophosphoglycan Exploits Binding to Galectin-1 and -3 to Modulate Epithelial Immunity. J Biol Chem. 2016; 291(2): 998–1013. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRyan CM, Mehlert A, Richardson JM, et al.: Chemical structure of Trichomonas vaginalis surface lipoglycan: a role for short galactose (β1-4/3) N-acetylglucosamine repeats in host cell interaction. J Biol Chem. 2011; 286(47): 40494–40508. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nde Miguel N, Lustig G, Twu O, et al.: Proteome analysis of the surface of Trichomonas vaginalis reveals novel proteins and strain-dependent differential expression. Mol Cell Proteomics. 2010; 9(7): 1554–1566. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nNoël CJ, Diaz N, Sicheritz-Ponten T, et al.: Trichomonas vaginalis vast BspA-like gene family: evidence for functional diversity from structural organisation and transcriptomics. BMC Genomics. 2010; 11: 99. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTwu O, de Miguel N, Lustig G, et al.: Trichomonas vaginalis exosomes deliver cargo to host cells and mediate host:parasite interactions. PLoS Pathog. 2013; 9(7): e1003482. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nLama A, Kucknoor A, Mundodi V, et al.: Glyceraldehyde-3-phosphate dehydrogenase is a surface-associated, fibronectin-binding protein of Trichomonas vaginalis. Infect Immun. 2009; 77(7): 2703–2711. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMundodi V, Kucknoor AS, Alderete JF: Immunogenic and plasminogen-binding surface-associated alpha-enolase of Trichomonas vaginalis. Infect Immun. 2008; 76(2): 523–531. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMundodi V, Kucknoor AS, Alderete JF: Antisense RNA decreases AP33 gene expression and cytoadherence by T. vaginalis. BMC Microbiol. 2007; 7: 64. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMa L, Meng Q, Cheng W, et al.: Involvement of the GP63 protease in infection of Trichomonas vaginalis. Parasitol Res. 2011; 109(1): 71–79. PubMed Abstract \| Publisher Full Text\n\nGould SB, Woehle C, Kusdian G, et al.: Deep sequencing of Trichomonas vaginalis during the early infection of vaginal epithelial cells and amoeboid transition. Int J Parasitol. 2013; 43(9): 707–719. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nLustig G, Ryan CM, Secor WE, et al.: Trichomonas vaginalis contact-dependent cytolysis of epithelial cells. Infect Immun. 2013; 81(5): 1411–1419. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nQuan JH, Kang BH, Cha GH, et al.: Trichonomas vaginalis metalloproteinase induces apoptosis of SiHa cells through disrupting the Mcl-1/Bim and Bcl-xL/Bim complexes. PLoS One. 2014; 9(10): e110659. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nQuintas-Granados LI, Villalpando JL, Vázquez-Carrillo LI, et al.: TvMP50 is an immunogenic metalloproteinase during male trichomoniasis. Mol Cell Proteomics. 2013; 12(7): 1953–1964. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCárdenas-Guerra RE, Arroyo R, Rosa de Andrade I, et al.: The iron-induced cysteine proteinase TvCP4 plays a key role in Trichomonas vaginalis haemolysis. Microbes Infect. 2013; 15(13): 958–968. PubMed Abstract \| Publisher Full Text\n\nCárdenas-Guerra RE, Ortega-López J, Flores-Pucheta CI, et al.: The recombinant prepro region of TvCP4 is an inhibitor of cathepsin L-like cysteine proteinases of Trichomonas vaginalis that inhibits trichomonal haemolysis. Int J Biochem Cell Biol. 2015; 59: 73–83. PubMed Abstract \| Publisher Full Text\n\nPuente-Rivera J, Ramón-Luing Lde L, Figueroa-Angulo EE, et al.: Trichocystatin-2 (TC-2): an endogenous inhibitor of cysteine proteinases in Trichomonas vaginalis is associated with TvCP39. Int J Biochem Cell Biol. 2014; 54: 255–265. PubMed Abstract \| Publisher Full Text\n\nRiestra AM, Gandhi S, Sweredoski MJ, et al.: A Trichomonas vaginalis Rhomboid Protease and Its Substrate Modulate Parasite Attachment and Cytolysis of Host Cells. PLoS Pathog. 2015; 11(12): e1005294. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEscobedo-Guajardo BL, González-Salazar F, Palacios-Corona R, et al.: Trichomonas vaginalis acidic phospholipase A2: isolation and partial amino acid sequence. Acta Parasitol. 2013; 58(4): 519–526. PubMed Abstract \| Publisher Full Text\n\nTwu O, Dessí D, Vu A, et al.: Trichomonas vaginalis homolog of macrophage migration inhibitory factor induces prostate cell growth, invasiveness, and inflammatory responses. Proc Natl Acad Sci U S A. 2014; 111(22): 8179–8184. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nde Miguel N, Riestra A, Johnson PJ: Reversible association of tetraspanin with Trichomonas vaginalis flagella upon adherence to host cells. Cell Microbiol. 2012; 14(12): 1797–1807. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nCoceres VM, Alonso AM, Nievas YR, et al.: The C-terminal tail of tetraspanin proteins regulates their intracellular distribution in the parasite Trichomonas vaginalis. Cell Microbiol. 2015; 17(8): 1217–1229. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nKusdian G, Woehle C, Martin WF, et al.: The actin-based machinery of Trichomonas vaginalis mediates flagellate-amoeboid transition and migration across host tissue. Cell Microbiol. 2013; 15(10): 1707–1721. PubMed Abstract \| Publisher Full Text\n\nFiori PL, Diaz N, Cocco AR, et al.: Association of Trichomonas vaginalis with its symbiont Mycoplasma hominis synergistically upregulates the in vitro proinflammatory response of human monocytes. Sex Transm Infect. 2013; 89(6): 449–454. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nFichorova RN, Lee Y, Yamamoto HS, et al.: Endobiont viruses sensed by the human host - beyond conventional antiparasitic therapy. PLoS One. 2012; 7(11): e48418. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nHuang KY, Chen YY, Fang YK, et al.: Adaptive responses to glucose restriction enhance cell survival, antioxidant capability, and autophagy of the protozoan parasite Trichomonas vaginalis. Biochim Biophys Acta. 2014; 1840(1): 53–64. PubMed Abstract \| Publisher Full Text\n\nLeitsch D, Kolarich D, Duchêne M: The flavin inhibitor diphenyleneiodonium renders Trichomonas vaginalis resistant to metronidazole, inhibits thioredoxin reductase and flavin reductase, and shuts off hydrogenosomal enzymatic pathways. Mol Biochem Parasitol. 2010; 171(1): 17–24. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nYeh YM, Huang KY, Richie Gan RC, et al.: Phosphoproteome profiling of the sexually transmitted pathogen Trichomonas vaginalis. J Microbiol Immunol Infect. 2013; 46(5): 366–373. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nHirt RP, de Miguel N, Nakjang S, et al.: Trichomonas vaginalis pathobiology new insights from the genome sequence. Adv Parasitol. 2011; 77: 87–140. PubMed Abstract \| Publisher Full Text\n\nPaschinger K, Hykollari A, Razzazi-Fazeli E, et al.: The N-glycans of Trichomonas vaginalis contain variable core and antennal modifications. Glycobiology. 2012; 22(2): 300–313. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRosenberger AF, Hangelmann L, Hofinger A, et al.: UDP-xylose and UDP-galactose synthesis in Trichomonas vaginalis. Mol Biochem Parasitol. 2012; 181(1): 53–56. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChatterjee A, Ratner DM, Ryan CM, et al.: Anti-Retroviral Lectins Have Modest Effects on Adherence of Trichomonas vaginalis to Epithelial Cells In Vitro and on Recovery of Tritrichomonas foetus in a Mouse Vaginal Model. PLoS One. 2015; 10(8): e0135340. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRada P, Doležal P, Jedelský PL, et al.: The core components of organelle biogenesis and membrane transport in the hydrogenosomes of Trichomonas vaginalis. PLoS One. 2011; 6(9): e24428. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nWexler-Cohen Y, Stevens GC, Barnoy E, et al.: A dynamin-related protein contributes to Trichomonas vaginalis hydrogenosomal fission. FASEB J. 2014; 28(3): 1113–1121. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nSchneider RE, Brown MT, Shiflett AM, et al.: The Trichomonas vaginalis hydrogenosome proteome is highly reduced relative to mitochondria, yet complex compared with mitosomes. Int J Parasitol. 2011; 41(13–14): 1421–1434. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBeltrán NC, Horváthová L, Jedelský PL, et al.: Iron-induced changes in the proteome of Trichomonas vaginalis hydrogenosomes. PLoS One. 2013; 8(5): e65148. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHorváthová L, Šafaríková L, Basler M, et al.: Transcriptomic identification of iron-regulated and iron-independent gene copies within the heavily duplicated Trichomonas vaginalis genome. Genome Biol Evol. 2012; 4(10): 1017–1029. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGarg S, Stölting J, Zimorski V, et al.: Conservation of Transit Peptide-Independent Protein Import into the Mitochondrial and Hydrogenosomal Matrix. Genome Biol Evol. 2015; 7(9): 2716–2726. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRada P, Makki AR, Zimorski V, et al.: N-Terminal Presequence-Independent Import of Phosphofructokinase into Hydrogenosomes of Trichomonas vaginalis. Eukaryot Cell. 2015; 14(12): 1264–1275. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBurstein D, Gould SB, Zimorski V, et al.: A machine learning approach to identify hydrogenosomal proteins in Trichomonas vaginalis. Eukaryot Cell. 2012; 11(2): 217–228. PubMed Abstract \| Free Full Text\n\nBrás XP, Zimorski V, Bolte K, et al.: Knockout of the abundant Trichomonas vaginalis hydrogenosomal membrane protein TvHMP23 increases hydrogenosome size but induces no compensatory up-regulation of paralogous copies. FEBS Lett. 2013; 587(9): 1333–1339. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nMorada M, Smid O, Hampl V, et al.: Hydrogenosome-localization of arginine deiminase in Trichomonas vaginalis. Mol Biochem Parasitol. 2011; 176(1): 51–54. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmith A, Johnson P: Gene expression in the unicellular eukaryote Trichomonas vaginalis. Res Microbiol. 2011; 162(6): 646–654. PubMed Abstract \| Publisher Full Text\n\nBradic M, Warring SD, Low V, et al.: The Tc1/mariner transposable element family shapes genetic variation and gene expression in the protist Trichomonas vaginalis. Mob DNA. 2014; 5: 12. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nHuang PJ, Lin WC, Chen SC, et al.: Identification of putative miRNAs from the deep-branching unicellular flagellates. Genomics. 2012; 99(2): 101–107. PubMed Abstract \| Publisher Full Text\n\nCui J, Das S, Smith TF, et al.: Trichomonas transmembrane cyclases result from massive gene duplication and concomitant development of pseudogenes. PLoS Negl Trop Dis. 2010; 4(8): e782. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWoehle C, Kusdian G, Radine C, et al.: The parasite Trichomonas vaginalis expresses thousands of pseudogenes and long non-coding RNAs independently from functional neighbouring genes. BMC Genomics. 2014; 15: 906. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nJiang I, Tsai CK, Chen SC, et al.: Molecular basis of the recognition of the ap65-1 gene transcription promoter elements by a Myb protein from the protozoan parasite Trichomonas vaginalis. Nucleic Acids Res. 2011; 39(20): 8992–9008. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWei SY, Lou YC, Tsai JY, et al.: Structure of the Trichomonas vaginalis Myb3 DNA-binding domain bound to a promoter sequence reveals a unique C-terminal β-hairpin conformation. Nucleic Acids Res. 2012; 40(1): 449–460. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChu CH, Chang LC, Hsu HM, et al.: A highly organized structure mediating nuclear localization of a Myb2 transcription factor in the protozoan parasite Trichomonas vaginalis. Eukaryot Cell. 2011; 10(12): 1607–1617. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nHsu HM, Chu CH, Wang YT, et al.: Regulation of nuclear translocation of the Myb1 transcription factor by TvCyclophilin 1 in the protozoan parasite Trichomonas vaginalis. J Biol Chem. 2014; 289(27): 19120–19136. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nHsu HM, Lee Y, Hsu PH, et al.: Signal transduction triggered by iron to induce the nuclear importation of a Myb3 transcription factor in the parasitic protozoan Trichomonas vaginalis. J Biol Chem. 2014; 289(42): 29334–29349. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nSmith AJ, Chudnovsky L, Simoes-Barbosa A, et al.: Novel core promoter elements and a cognate transcription factor in the divergent unicellular eukaryote Trichomonas vaginalis. Mol Cell Biol. 2011; 31(7): 1444–1458. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFuentes V, Barrera G, Sánchez J, et al.: Functional analysis of sequence motifs involved in the polyadenylation of Trichomonas vaginalis mRNAs. Eukaryot Cell. 2012; 11(6): 725–734. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSimoes-Barbosa A, Hirt RP, Johnson PJ: A metazoan/plant-like capping enzyme and cap modified nucleotides in the unicellular eukaryote Trichomonas vaginalis. PLoS Pathog. 2010; 6(7): e1000999. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "12370", "date": "11 Feb 2016", "name": "Robert Hirt", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12371", "date": "11 Feb 2016", "name": "Sven B. Gould", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-162
https://f1000research.com/articles/5-161/v1	11 Feb 16	{ "type": "Review", "title": "Recent advances in the understanding and management of IgA nephropathy", "authors": [ "Kar Neng Lai", "Joseph C.K. Leung", "Sydney C.W. Tang", "Joseph C.K. Leung", "Sydney C.W. Tang" ], "abstract": "Since its first description in 1968, IgA nephropathy has remained the most common form of primary glomerulonephritis leading to chronic kidney disease in developed countries. The clinical progression varies, and consequent end-stage renal disease occurs in 30% to 40% of patients 20 to 30 years after the first clinical presentation. Current data implicate overproduction of aberrantly glycosylated IgA1 as being pivotal in the induction of renal injury. Effective and specific treatment is still lacking, and new therapeutic approaches will be developed after better understanding the disease pathogenesis.", "keywords": [ "IgA nephropathy", "glomerulonephritis", "end-stage renal disease", "genetics of IgAN", "Familial IgAN", "treatment IgAn" ], "content": "Introduction\n\nIgA nephropathy (IgAN) remains the most common primary glomerulonephritis worldwide. Other than diabetic nephropathy, IgAN remains the next important health-care issue in nephrology as it often affects young adults and the nephropathy pursues a slow but relentless clinical course. Consequent end-stage renal disease (ESRD) occurs in 30% to 40% of patients within 20 to 30 years after clinical presentation. The kidney is a target of injury in IgAN, yet the primary defect originates from a systemic aberrant glycosylation of O-linked glycans in the hinge region of IgA1, resulting in increased serum levels of galactose-deficient IgA1 (Gd-IgA1). As the immunochemical abnormality of IgA is not corrected by renal transplantation, not surprisingly IgAN can frequently recur in allograft. Effective and specific treatment for IgAN is still lacking.\n\nSerum Gd-IgA1 levels are heritable in a dominant pattern with reduced penetrance, although most patients’ relatives who have high serum levels of Gd-IgA1 do not exhibit clinical manifestations of renal injury. IgAN may occur in either sporadic or familial pattern. Familial IgAN may have a poorer prognosis with an increased risk of progression to renal failure, but this is controversial. Patients with familial IgAN have increased serum levels of galactose-deficient macromolecular IgA1 as compared with patients with sporadic IgAN. Macromolecular IgA1 isolated from patients with familial IgAN has enhanced binding to mesangial cells in vitro. These observations support the notion that genetic factors are involved in the pathogenesis of familial, as well as sporadic, IgAN, and risk factors of multiple candidate genes have been identified in different ethnic groups. The goal of this review is to present the genetic data discovered in the last decade, and discuss the treatment options in IgAN.\n\n\nGenetic data\n\nEpidemiological data support a strong genetic contribution to IgAN1. First, there are significant geographic and ethnic differences in the prevalence of IgAN; the highest frequency is in East Asians, and it is relatively common in Mediterranean countries and very uncommon in individuals of African ancestry2,3. Second, IgA1 glycosylation defects exhibit high heritability in relatives of familial IgAN patients4–6. Familial IgAN presenting as autosomal dominant transmission with incomplete penetrance has been well recognized1. Despite numerous efforts at gene mapping by using linkage approaches, Mendelian defects responsible for familial IgAN remain elusive unless large families are available for study. Earlier studies of IgAN families revealed four linked loci.\n\n6q22-q23 (IGAN1) and 3p24-p23 loci were the first loci identified in linkage analysis of IgAN with a logarithm of the ratio of odds (LOD) score of 5.67. The study analyzed 24 Italian and six American families, suggesting another locus at 3p24-p23 with a maximum LOD score of 2.8. Next, 2q36 locus was revealed in a four-generation Canadian family of German-Austrian origin with 14 affected and 11 unaffected members8. The pedigree is consistent with autosomal dominant inheritance. Parametric and non-parametric linkage analysis produced significant LOD scores according to standard criteria for Mendelian disease. Finally, a European IgAN consortium identified suggestive loci at 4q26-q31 (LOD score of 1.8) and 17q12-q22 (LOD score of 2.6) in 22 Italian families with 59 affected and 127 unaffected members. The loci were named IGAN2 and IGAN9. Intriguingly, these four loci have not been revealed in familial IgAN of other ethnicity10.\n\nThe genome-wide association study (GWAS) approach has emerged as a powerful alternative to family-based studies for complex traits and has been successfully applied to IgAN. The first GWAS for IgAN based on European patients11 was shortly followed by two larger studies performed in Chinese cohorts of Hans ethnicity12,13. Notably, all three studies consisted of a relatively small discovery sample of 3,000 IgAN cases. Seven independent risk loci with genome-wide significance (P < 5 × 10-8) were identified and these loci cumulatively explained approximately 5% of the overall disease variance.\n\nThe IgAN risk allele frequencies correlate well with disease epidemiology3. Notably, East Asians with the highest prevalence of IgAN carry the highest average number of risk alleles, but IgAN is less common in Africans with the lowest burden of risk alleles. IgAN as a leading cause of ESRD is nearly 10-fold higher among US kidney patients with East Asian ancestry when compared with African Americans3. Since over 85% of the existing GWAS discovery cohorts are ethnic Chinese, studies in other ethnicities (especially from Mediterranean countries and Australasia with moderate prevalence) are needed to understand genetic risk profiles among other populations.\n\nContrary to the genetic approaches using linkage studies, and GWAS that requires a large patient cohort of sporadic IgAN, Liu et al.14 studied 10 IgAN families of Han Chinese ethnicity by using exome sequencing techniques. IgAN families are enriched in genetic components predisposing individuals to the development of this disorder. The technique of exome sequencing allows the interrogation of the whole exome to identify genes and gene variants that underlie both monogenic and complex diseases. Six deleterious variants in four genes associated with familial IgAN were discovered. Of interest is the association of DEFA gene and the disease susceptibility in both sporadic and familial IgAN of Han Chinese ethnicity with different mutations14,15.\n\nThrough careful analysis and annotation of the detected loci, several causal candidate genes have been prioritized, linking pathways involved in the pathogenesis of IgAN. The implicated pathways include (i) the antigen-processing and presentation pathway (three loci on chromosome 6p21 in the major histocompatibility complex [MHC] region), (ii) the mucosal immunity pathway (chromosomes 22q12 HORMAD2 locus, 8p23 α-defensin [DEFA] locus, and 17p13 TNFSF13 locus), and (iii) the alternative complement pathway (chromosome 1q32 complement factor H [CFH] locus)11–13.\n\nAll three GWASs of IgAN identified strong signals within the MHC region with three distinct susceptibility loci on chromosome 6p21: HLA-DRB1/DQB1, HLA-DPB1/DPB2, and TAP1/PSMB9. HLA-DRB1-DQA1 and -DQB1 genes carry the strongest association, and the DQB10602-DRB11501 haplotype confers a highly protective effect12.\n\nThe second distinct MHC locus was centered over the region of the HLA-DPA1, -DPB1, and -DPB2 genes (also encoding MHC-II molecules), but the causal variant at this locus and its involvement in IgAN are still not known. The third MHC locus contained the TAP1, TAP2, PSMB8, and PSMB9 genes. These genes play an important role in modulation of cytokine production and cytotoxic T-cell response through antigen processing for presentation by MHC-I molecules.\n\nThe clinical characteristic of synpharyngitic macroscopic hematuria led to the hypothesis that defects in the regulation of local IgA response or abnormal mucosal antigen handling (or both) may trigger IgAN16.\n\nAPRIL, a proliferation-inducing ligand, is the molecule involved in T cell-independent generation of IgA-secreting plasma cells as well as in the IgA1 to IgA2 class switching. A GWAS locus on chromosome 17p13 contains TNFSF13 that encodes APRIL. Serum levels of APRIL are elevated in some patients with IgAN17, and raised total serum IgA occurs with the 17p23 risk variant12. Overexpression of B-cell activation factor (BAFF), a related molecule with overlapping functions and receptors with APRIL, results in mesangial IgA deposits in mice17. A recent study from Japan showed that treatment of the newly developed grouped ddY (gddY) mice with anti-APRIL antibody reduced serum IgA levels, glomerular IgA deposition, albuminuria, and renal damage18. These data suggest that APRIL and BAFF signaling may be involved in the pathogenesis of IgAN and that both may be potential therapeutic targets.\n\nA locus on chromosome 22q12 also influences serum IgA levels and encompasses several genes, including the IL-6 family-encoding genes LIF and OSM11.\n\nThe DEFA gene cluster on chromosome 8p23 is the third IgAN GWAS locus implicated in mucosal immunity. The α-defensin gene family encodes small, structurally related peptides that are secreted at mucosal surfaces with microbiocidal and chemoattractant properties19. α-defensin 1, 3, and 4 (encoded by DEFA1, DEFA3, and DEFA4) are synthesized in neutrophils, whereas α-defensin 5 and 6 (DEFA5 and DEFA6) are constitutively released by the intestinal Paneth cells into the gut lumen. It remains unclear whether the IgAN risk allele in this region confers a risk haplotype due to excessive copies of DEFA1/3 genes or variants of DEFA5/6 genes.\n\nLately, six new genome-wide significant associations—four in ITGAM, ITGAX, VAV3, and CARD9 and two new independent signals at HLA-DQB1 and DEFA—were identified in a GWAS examining 20,612 IgAN individuals of European and East Asian ancestry20. Most loci are directly associated with either risk of inflammatory bowel disease or maintenance of the intestinal epithelial barrier and response to mucosal pathogens. A possible role for host-intestinal pathogen interactions in shaping the genetic landscape of IgAN has been proposed.\n\nA common deletion (deleting CFHR3 and CFHR1 genes), within the CFH locus on chromosome 1q32, was found to be protective against IgAN in a GWAS studying both European and Asian populations12. The CFH gene encodes Factor H (FH) that regulates the alternative complement pathway. FH-related proteins (FHR1–5) are structurally similar to FH and are encoded by five genes (CFHR1-5) residing within the same genomic region. Given the high level of sequence similarity between CFH and CFHRs, these genes are believed to have originated through segmental duplications and are prone to recurrent structural rearrangements. CFHR3,1Δ is the most common variant; allelic frequency ranges from 0% to 5% in East Asians to 20% in Europeans and up to 50% in some African populations21. Each additional copy of CFHR3,1Δ reduces the risk of IgAN by approximately 40%12.\n\n\nTreatment\n\nPatients with minor urine abnormalities and normal blood pressure and glomerular filtration rate (GFR) usually do well and require only periodic monitoring, such as biennial clinic visits. For other patients, the therapeutic options are limited and include non-specific treatment to reduce proteinuria by renin-angiotensin system (RAS) blockade and non-specific control of inflammation using fish oil and agents such as corticosteroids, cytotoxic agents, anti-metabolite, and immunomodulatory drugs.\n\nRenin-angiotensin-aldosterone axis blockade. Evidence accumulated from 56 studies and 2,838 participants showed that only anti-hypertensive drugs—mostly angiotensin-converting enzyme inhibitor (ACEI) or angiotensin receptor blocker (ARB)—provided useful intervention mainly by reducing proteinuria22. RAS blockers are often prescribed for patients with IgAN and proteinuria. In a meta-analysis of 585 patients from 11 randomized clinical trials (RCTs)23, significant renoprotection and reduction of proteinuria were achieved with an ACEI or ARB versus control. The beneficial effects are promoted by concomitant dietary sodium and phosphate restriction. In addition, the efficacy of RAS blockade could be modified by ACE (I/D) gene polymorphisms such that, in the future, personalized medicine could be developed using pharmacogenomics data24.\n\nAliskiren is an oral direct renin inhibitor that has a theoretical basis for fully suppressing the RAS as ACEI or ARB treatment leads to a reactive increase in plasma renin activity. So far, the only two trials from Hong Kong showed an anti-proteinuric effect on top of ACEI or ARB therapy25,26. Patients with more advanced chronic kidney disease are prone to developing hyperkalemia. Long-term outcomes have not been reported.\n\nFish oil. The possible benefit of fish oil containing omega-3 polyunsaturated fatty acid in the treatment of IgAN rests on reducing intra-renal inflammation by mitigating inflammatory cytokines and eicosanoids. However, the published reports failed to show convincing benefits.\n\nIn the original Mayo Clinic multicenter study with 106 subjects27, fewer patients randomly assigned for fish oil treatment reached the end-point of at least a 50% rise in serum creatinine. Notably, neither this original study nor a subsequent trial showed a reduction of proteinuria. Proteinuria is a key therapeutic target because it may itself cause renal injury, and its reduction correlates with preservation of renal function. A recent trial of 30 patients suggested that a RAS blocker combined with polyunsaturated fatty acids reduced proteinuria more than RAS blocker alone28. The KDIGO (Kidney Disease: Improving Global Outcomes) 2012 Clinical Practice Guidelines29 suggest optional use of fish oil in the treatment of patients with persistent proteinuria of more than 1 g/day, despite 3 to 6 months of optimized supportive care including ACEI or ARBs and blood pressure control. Yet, the long-term benefits on preventing ESRD are uncertain.\n\nAs stated earlier, IgAN is an autoimmune kidney disease and hence immune modulation targeting the putative pathogenic pathways may alter the disease progression. To date, no medications have been approved by the US Food and Drug Administration specifically for IgAN. The availability of new agents with novel mechanisms and activities against the humoral immune response may allow targeted treatment. Herein, we examine the existing evidence for immunosuppressive therapy in IgAN.\n\nCorticosteroids. Since early 1980, corticosteroids were often prescribed to IgAN patients with moderate to severe persisting proteinuria (variably defined as more than 0.5 to 1.0 g/day lasting for at least 3 months). A meta-analysis of nine randomized controlled trials (including 536 patients with urinary protein excretion of more than 1 g/day and normal renal function) suggested that high-dose and short-term corticosteroid therapy produced significant renal protection but that low-dose, long-term corticosteroid use did not30. The 2012 KDIGO Guidelines29 recommend that patients with persistent proteinuria of more than 1 g/day despite adequate ACEI or ARB and blood pressure control and a GFR of more than 50 ml/min per 1.73 m2 receive a 6-month course of steroid therapy. A significant knowledge gap thus existed because patients with an estimated GFR (eGFR) of 30 to 50 ml/min per 1.73 m2 have been excluded from virtually all major clinical trials. A recent retrospective analysis of the European Validation Study of the Oxford Classification of IgAN (VALIGA) cohort of 1,147 patients (mostly white) may help to address this gap31. In the propensity score analysis, adding corticosteroid to RAS blocker resulted in a better reduction of proteinuria, a slower rate of renal function decline, and increased renal survival in comparison with administering RAS blocker alone in two groups of patients with a similar risk profile of progression. These benefits extended to 115 patients with an eGFR of less than 50 ml/min per 1.73 m2, and the benefits increased proportionally with the level of baseline proteinuria. However, the study is limited by its retrospective nature, unknown corticosteroid dosing regimens, frequent combination of corticosteroids with other immunosuppressive therapies, the potential for unmeasured and selection bias, and the potential for selection of patients by the participating center32.\n\nTwo new trials were conducted to further address the therapeutic value of conventional corticosteroid. STOP-IgAN is a German trial that randomly assigned adults with an eGFR of at least 30 ml/min per 1.73 m2 and persistent proteinuria of more than 0.75 g per day despite 6 months of supportive care (in particular, blockade of the RAS to a target blood pressure of less than 125/75 mm Hg) to receive supportive care alone or supportive care plus immunosuppression (corticosteroids alone if eGFR was 60 to 89 ml/min per 1.73 m2 or in combination with cyclophosphamide for the initial 3 months, followed by azathioprine if eGFR was 30 to 59 ml/min per 1.73 m2)33. During the run-in phase completed by 309 of 337 patients, proteinuria decreased to less than 0.75 g per day in 30% of the subjects, who then became ineligible for random assignment. Of 154 patients who underwent random assignment and completed 3 years of treatment, more patients in the immunosuppression group achieved full clinical remission (urine protein/creatinine of less than 0.2 g/g and reduction in eGFR of less than 5 ml/min per 1.73 m2), but there was no significant difference in the annual decline in eGFR between the two groups. Patients in the immunosuppression group had a significantly lower mean proteinuria level than those in the supportive-care group at 12 months after random assignment, but this difference disappeared at 36 months. The major conclusion was that the addition of immunosuppressive therapy to intensive supportive care did not significantly improve the outcome and may increase adverse effects. The study is limited by its open-label nature, relatively short duration of follow-up for the end-point of renal deterioration, the lack of histologic stratification for inclusion, and the questionable design of assigning steroid monotherapy to patients with an eGFR of more than 60 ml/min per 1.73 m2 and the addition of cyclophosphamide followed by azathioprine to patients with even lower eGFR. Finally, the findings could be conflicted by a relatively high proportion of subjects who were given combined ACEi and ARB treatment.\n\nTESTING is another large multicenter, double-blinded, randomized, placebo-controlled trial in progress. TESTING started recruitment in 2012 internationally to investigate the efficacy of oral methylprednisolone versus placebo in IgAN. The study includes patients with a wide range of eGFR values, from 20 to 90 ml/min per 1.73 m2.\n\nCyclophosphamide in combination with corticosteroids. In Caucasian subjects, cyclophosphamide plus corticosteroid therapy may benefit patients at high risk of developing ESRD, namely those with glomerular crescents and rapidly progressive clinical course34,35. In Chinese patients, crescentic IgAN carries a poor prognosis. Amongst 113 such patients from eight centers across China, no benefit was observed in the renal survival when cyclophosphamide was added to pulse corticosteroid therapy36. The 2012 KDIGO Guidelines29 suggest the use of corticosteroids and cyclophosphamide in patients with IgAN and rapidly progressive crescentic IgAN, analogous to the treatment of anti-neutrophil cytoplasmic antibody (ANCA) vasculitis, but this is not based on evidence from clinical trials.\n\nTonsillectomy in combination with corticosteroids. The practice of tonsillectomy in IgAN (mainly in Japan) is based on observation of disease activation, manifested as macroscopic hematuira and renal dysfunction following upper respiratory tract infection. Recently, a gene encoding glycosyltransferase involved in glycosylation of O-glycan in IgA molecules has been demonstrated to be downregulated in tonsillar B lymphocytes from patients with IgAN37. Outside Japan, the benefits of tonsillectomy have not been documented. A meta-analysis of seven non-randomized studies (mostly from Japan) comprising 858 patients (534 underwent tonsillectomy and 324 did not) showed that tonsillectomy combined with either standard or pulse corticosteroid treatment, but not tonsillectomy or corticosteroid treatment alone, resulted in higher remission rates with favorable long-term outcome38. Lately, a multicenter randomized controlled trial from Japan showed that tonsillectomy combined with steroid pulse therapy has no beneficial effect over steroid pulses alone to attenuate hematuria and to increase the incidence of clinical remission39. Although the anti-proteinuric effect was greater in combined therapy, the difference was marginal. Finally, a recent meta-analysis of 14 studies (also mostly from Japan) also found positive effects of tonsillectomy plus pulse or conventional steroid40. The 2012 KDIGO Guidelines29 suggest that tonsillectomy not be performed for IgAN.\n\nAzathioprine. A recent moderately large-scale study randomly assigned 207 patients to either corticosteroids alone (n = 106) or in combination with azathioprine (n = 101) for 6 months41. Azathioprine conferred no additional benefit but resulted in more adverse events, namely hepatotoxicity, anemia, and gastrointestinal symptoms. The 2012 KDIGO Guidelines29 do not recommend the use of azathioprine in IgAN.\n\nMycophenolate mofetil. Three studies in Chinese patients showed a benefit of mycophenolate mofetil (MMF): (i) In 62 patients with severe IgAN and urinary protein of more than 2 g/day, the MMF group showed significant improvement in proteinuria and serum lipids than the prednisone group42. (ii) Among 40 Chinese patients with mild tubulointerstitial lesions with persistent proteinuria of more than 1 g/day despite full RAS blockade, MMF treatment for 6 months resulted in significant reduction in proteinuria43 and improved renal survival at 6-year follow-up44. (iii) In a study comparing therapy with MMF/prednisone and cyclophosphamide/prednisone for severe IgAN45, the former regimen achieved a higher remission rate with better reduction of proteinuria and improvement of renal function and less adverse effects.\n\nIn contrast, three studies in Caucasians showed mixed results: (i) Among 34 Belgian patients with impaired renal function, histologic unfavorable criteria and arterial hypertension, a combination of salt restriction, ACEI therapy and high-dose MMF failed to demonstrate a better beneficial effect after 3 years of evaluation46. (ii) In an American study that recruited patients with even more advanced renal insufficiency, a worse outcome occurred with MMF as a “salvage” therapy47. (iii) In another Italian study, a subset of IgAN patients with florid glomerular changes treated with MMF and corticosteroids showed remission of proteinuria and reversal of progressive renal failure48.\n\nGiven that these mixed results occurred across different ethnic groups and that none of these studies was adequately powered to provide a definitive answer, the 2012 KDIGO Guidelines29 suggest not using MMF in IgAN. More recently, one trial conducted in 52 children, adolescents, and adults with IgAN in the US and Canada was terminated prematurely as MMF did not reduce proteinuria49. Patients received lisinopril (or losartan) plus a highly purified omega-3 fatty acid for 3 months during run-in, and only those with a persistent urinary albumin/creatinine ratio of at least 0.6 g/g (males) or at least 0.8 g/g (females) were randomly assigned.\n\nIncreased knowledge on the pathogenetic mechanisms of IgAN, particularly on the role of mucosal immunity and B-cell activation, has provided the impetus for several new phase II/III clinical trials. None of these have reached any conclusions yet.\n\nEnteric budesonide. NEFIGAN is a phase IIb trial that was started in 2012 to evaluate the efficacy and safety of an enteric budesonide delivered specifically to the ileocecal Peyer’s patches in primary IgAN across 10 European countries. Preliminary study demonstrated a reduction of proteinuria by 23% and a modest improvement of eGFR by 8% in 16 patients (proteinuria of more than 0.5 g/day and serum creatinine of less than 200 μmol/l) after 6 months of enteric budesonide, followed by 3 months of further observation50. The study was completed in September 2015 and found encouraging results in terms of proteinuria reduction and stabilization of renal function at 9 months (ASN Kidney Week 2015).\n\nB-cell depletion/inhibition. Blisibimod is a selective peptibody antagonist of BAFF that can be administered subcutaneously. BRIGHT-SC (Blisibimod Response in IgAN Following At-Home Treatment by Subcutaneous Administration) began in 2013 and is currently recruiting patients in Asia and Europe.\n\nSpleen tyrosine kinase (Syk) inhibition. An important molecule within an intracellular signaling pathway activated upon ligation of the B-cell receptor is Syk, which mediates maturation and survival of the B-cell lineage. Pharmacological inhibition of Syk, or its knockdown by small interfering RNA (siRNA), significantly reduced cellular proliferation and the synthesis of pro-inflammatory mediators in human mesangial cells exposed to IgA1 from patients with IgAN51. SIGN (Syk Inhibition for Glomerulonephritis), a multicenter trial, started recruitment in 2014 globally to evaluate the efficacy of fostamatinib (a selective oral Syk inhibitor) in patients with IgAN.\n\nProteasomal inhibition. There is preliminary evidence for a role of increased immunoproteasome activity in IgAN52. A single-center, open-label, exploratory study examining the effects of bortezomib (Velcade) in IgAN was started in 2010 in the US.\n\n\nConclusions\n\nDespite a better understanding of the immunochemical nature of aberrantly glycosylated IgA1 and the genetic risk profile of IgAN, the key issue of how disease can be triggered following recurrent mucosal infection remains unknown. Specific treatment is lacking. Diagnosis by a non-invasive method such as disease biomarkers without invasive renal biopsy will allow increased disease detection rate and early treatment intervention.", "appendix": "Competing interests\n\n\n\nThe authors declare that they 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\nKiryluk K, Julian BA, Wyatt RJ, et al.: Genetic studies of IgA nephropathy: past, present, and future. Pediatr Nephrol. 2010; 25(11): 2257–68. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHall YN, Fuentes EF, Chertow GM, et al.: Race/ethnicity and disease severity in IgA nephropathy. BMC Nephrol. 2004; 5: 10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKiryluk K, Li Y, Sanna-Cherchi S, et al.: Geographic differences in genetic susceptibility to IgA nephropathy: GWAS replication study and geospatial risk analysis. PLoS Genet. 2012; 8(6): e1002765. 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PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nLiu X, Dewei D, Sun S, et al.: Treatment of severe IgA nephropathy: mycophenolate mofetil/prednisone compared to cyclophosphamide/prednisone. Int J Clin Pharmacol Ther. 2014; 52(2): 95–102. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nMaes BD, Oyen R, Claes K, et al.: Mycophenolate mofetil in IgA nephropathy: results of a 3-year prospective placebo-controlled randomized study. Kidney Int. 2004; 65(5): 1842–9. PubMed Abstract \| Publisher Full Text\n\nFrisch G, Lin J, Rosenstock J, et al.: Mycophenolate mofetil (MMF) vs placebo in patients with moderately advanced IgA nephropathy: a double-blind randomized controlled trial. Nephrol Dial Transplant. 2005; 20(10): 2139–45. PubMed Abstract \| Publisher Full Text\n\nRoccatello D, Rossi D, Marletto F, et al.: Long-term effects of methylprednisolone pulses and mycophenolate mofetil in IgA nephropathy patients at risk of progression. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "12368", "date": "11 Feb 2016", "name": "Todd Ing", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12369", "date": "11 Feb 2016", "name": "Keng-Thye Woo", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12367", "date": "11 Feb 2016", "name": "Yusuke Suzuki", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-161
https://f1000research.com/articles/5-160/v1	11 Feb 16	{ "type": "Research Article", "title": "Patterns of database citation in articles and patents indicate long-term scientific and industry value of biological data resources", "authors": [ "David Bousfield", "Johanna McEntyre", "Sameer Velankar", "George Papadatos", "Alex Bateman", "Guy Cochrane", "Jee-Hyub Kim", "Florian Graef", "Vid Vartak", "Blaise Alako", "Niklas Blomberg", "David Bousfield", "Johanna McEntyre", "Sameer Velankar", "George Papadatos", "Alex Bateman", "Guy Cochrane", "Jee-Hyub Kim", "Florian Graef", "Vid Vartak", "Blaise Alako" ], "abstract": "Data from open access biomolecular data resources, such as the European Nucleotide Archive and the Protein Data Bank are extensively reused within life science research for comparative studies, method development and to derive new scientific insights. Indicators that estimate the extent and utility of such secondary use of research data need to reflect this complex and highly variable data usage. By linking open access scientific literature, via Europe PubMedCentral, to the metadata in biological data resources we separate data citations associated with a deposition statement from citations that capture the subsequent, long-term, reuse of data in academia and industry. We extend this analysis to begin to investigate citations of biomolecular resources in patent documents. We find citations in more than 8,000 patents from 2014, demonstrating substantial use and an important role for data resources in defining biological concepts in granted patents to both academic and industrial innovators. Combined together our results indicate that the citation patterns in biomedical literature and patents vary, not only due to citation practice but also according to the data resource cited. The results guard against the use of simple metrics such as citation counts and show that indicators of data use must not only take into account citations within the biomedical literature but also include reuse of data in industry and other parts of society by including patents and other scientific and technical documents such as guidelines, reports and grant applications.", "keywords": [ "Data citations", "Data reuse", "Data repositories", "Data archiving", "Open data", "Bibliometrics", "Patent analysis", "Research impact" ], "content": "Introduction\n\nOpen sharing of data is a well-established norm in molecular biology and the genomic sciences: protein structure datasets are released to the community after the corresponding articles are published, many genome sequencing projects deposit sequences in public archives as soon as they are acquired. Consequently, the bioinformatics databases holding these data1 form an essential part of molecular biology research. The standardisation, organisation and careful annotation that occurs when experimental data is deposited in openly accessible biomolecular resources such as the European Nucleotide Archive2 or the Protein Data Bank3 enables independent data verification and also support and encourage data reuse by the research community. The deposition of experimental data in structured archives is complemented by a long tradition of manual curation in which protein properties, biological reactions, genetic linkages and other facts from the scientific literature are further catalogued into structured reference collections such as UniProt4, RefSeq5, and OMIM6. Value-adding data resources build on, and further combine, this treasure-trove of open data and provide comprehensive coverage of biology by cataloguing model organisms, protein classes, sequence motifs, biological pathways, reactions, metabolites: to date over 1600 biological databases are reported in the Nucleic Acids Research database catalogue7.\n\nMaintaining and updating an infrastructure to support the active collection, annotation and redistribution of data is costly and only makes sense if there is a research community that actively reuses the data. While the value of opening up data for independent validation is seen as imperative for the scientific debate8, the open datasets from molecular biology research have long been used to stimulate and test additional hypotheses that are independent of the original experiment. The aggregation and inter-linking of published datasets also forms the basis for meta-analysis, modelling or new derivative databases9–13. Hence, managing these resources in an effective and sustainable manner requires database owners and funders to understand their usage and role in scientific research, as well as their role in generation of downstream societal value, for example by contributing to the definition of intellectual property held in patent documents. Quantitative analysis of data citation in scientific articles currently lacks metrics that parallel traditional scientific article citation indices and journal impact factors. Furthermore publishers of scientific journals rarely annotate database citations leaving organisations such as Europe PubMed Central (EPMC) to provide routine text-mining to find citations of database identifiers in full-text articles14.\n\nEstimating the on-going use of biological data resources by means of their citation patterns in scientific articles captures one aspect of data reuse but is challenging because data citations in the scientific literature are highly variable with few established community norms. For example, Piwowar and colleagues15 tracked the citation practices used by three life science data resources: NCBI’s GEO16, Pangaea17, and Treebase18. They manually curated data citation statements in a corpus of data-citing papers and noted that for datasets from Pangaea the norm was data citation via the reference list while for the other resources a significant proportion of the citations were made by direct mention of the unique data resource identifier in the text narrative. This variable citation practice, and the subsequent problem this poses for estimations of data usage by tracking data citations in the literature is further exemplified by Belter in a study of the data citation practices used by oceanographers19. Despite the fact that the datasets studied had unambiguous terms of use, including recommendations for citation, the citation practices observed were highly variable with most citations occurring as a direct reference in the main text of the journal article. For example, Belter found that the editions of the National Oceanographic Data Center climate data set were cited in no less than 1180 different ways within his curated literature corpus.\n\nDespite these challenges Kafkas et al.14 have shown that text-mining database citation identifiers, i.e. the juxtaposition in the text of a database name and an appropriate accession ID, from an Open Access literature corpus within EPMC doubled the number of data citations compared to the number supplied by publishers. Subsequently they extended their study with an analysis of the supplementary material associated with the same corpus and noted that data citation practices in supplementary data files differed markedly from those observed in the main article20. For instance, supplementary files often contain long lists of database identifiers. The rank of databases when ordered by the frequency of data citations also differed in supplementary data files compared to that observed in the main articles from the same corpus.\n\nCollectively these studies give us a general sense of the scale of data use although the highly diverse citation practices observed cautions against a naive application of data citation as a metric for research impact. Furthermore, the statistics generated by the studies described above do not discriminate between citations arising from the initial deposition and publication of a source article and subsequent secondary citations in the research literature. Nor do these studies describe the flow and indirect use of data through the web of existing bioinformatics data resources. Thus there is a need to further investigate data citations to serve as a background for development of usage metrics, guide the life-cycle management of resources and understand the flow and impact of biological data. We build on and extend earlier studies by demonstrating how primary data citations, arising from the deposition of data and its citation in the source article describing the generation of the data, can be separated from subsequent secondary data citations. In this study we focussed our attention on two of the major biomolecular databases, the European Nucleotide Archive (ENA) and the Protein Data Bank (PDB), where the high-quality curation and well-established links between an open literature resource (EPMC) and data resources allow us to dissect primary from secondary data citations. We have done this by combining accession publication data from the biomolecular resources with the citation data from EPMC in order to provide an insight into dynamics of data citations over time. We further extend our study of data citations by mining a corpus of full-text patent documents (accessed via SureChEMBL21) in order to begin to understand the downstream use of data resources in the definition of biological entities and concepts in a legal/technical commercial environment.\n\n\nMethods\n\nThe full-text research articles used in this study were accessed from EPMC22. The content scope of EPMC covers over 25 million PubMed abstracts and 3.5 million full-text articles (see https://europepmc.org/About), each article is identified by a unique identifier (a “PMID” for abstracts and a “PMCID” for full-text). Data accession references were extracted using EPMC’s text-mining pipeline based on a combination of rule-based knowledge about possible accession number structures and an empirically-determined set of contextual cues14,20,23. The pipeline is integrated into the EPMC infrastructure (http://europepmc.org/) and is used to identify instances of data citation in full-text articles on a daily basis. The data citations are made publically available via EPMC’s APIs. When comparing research articles with patents we focused on 2014 as the most recent year available. However, due to the fact that embargoed articles were still being added at the time of our study, we repeated our analysis using material from 2012 and 2013 to ensure that our comparisons were robust.\n\nThe Protein Data Bank (PDB) is the global archive of 3D structures of proteins, nucleic acids and complex assemblies. This large corpus of data (94,117 holdings in 2014) and related citations provide an extensive test set for developing and understanding data citation and access metrics (http://www.wwpdb.org/stats/deposition). We used the European site PDBe as the definitive source of deposition data, i.e. accession identifiers, deposition dates and associated PMID publication details.\n\nThe European Nucleotide Archive (ENA; http://www.ebi.ac.uk/ena) is Europe's primary resource for nucleotide sequence information. The current size of the ENA is in excess of 2.5 petabytes, with a doubling time of approximately 20 months (see http://www.ebi.ac.uk/ena/about/statistics). We used ENA as an additional definitive source of deposition data, i.e. accession identifiers, deposition dates and associated PMID publication details.\n\nSureChEMBL (https://www.surechembl.org/) is a publicly available, large-scale resource containing chemical annotations found in the full-text, images and attachments of patent documents21. Its data content at 28 October 2015 included more than 14 million chemically annotated full-text patent documents. In addition, it contains 130 million patent abstracts from DOCDB, the European Patent Office master documentation database with worldwide coverage containing bibliographic data, abstracts, and citations (but no full-text or images).\n\nSureChEMBL provides full-text searching of the patent literature using a keyword-based querying functionality, complemented by a chemistry-based query engine. Our queries retrieved full-text patent documents (both applications and granted patents) written in the English language, published in 2014 by the three main patent authorities, namely the European Patent Office (EPO), the US Patent and Trademark Office (USPTO) and the World Intellectual Property Organisation (WIPO). To ensure the relevance of the retrieved patent documents to biological and life sciences, the appropriate international patent classification (IPC http://www.wipo.int/classifications/ipc/en/) codes (predominantly from categories A (human necessities) and C (chemistry), full query: “(ic:(A01 OR A23 OR A24 OR A61 OR A62B OR C05 OR C06 OR C07 OR C08 OR C09 OR C10 OR C11 OR C12 OR C13 OR C14 OR G01N) OR cpc:(A01 OR A23 OR A24 OR A61 OR A62B OR C05 OR C06 OR C07 OR C08 OR C09 OR C10 OR C11 OR C12 OR C13 OR C14 OR G01N) OR ecla_ec:(A01 OR A23 OR A24 OR A61 OR A62B OR C05 OR C06 OR C07 OR C08 OR C09 OR C10 OR C11 OR C12 OR C13 OR C14 OR G01N)) AND desc:the AND pdyear:[2010 TO 2014] AND pnlang:EN AND pnctry:(WO OR EP OR US)” ) were used to filter the results24. No further selection was carried out on the basis of patent kind (an indication of where the patent is in the review process, e.g. application stage, or granted). Patent families were identified using the simple patent family definition provided by the European Patent Office (EPO)25 and a single example selected at random to be sole representative of the group in subsequent analysis. In total, 188,589 documents published in 2014 were retrieved and used as input for the identifier extraction process. The XML content generated by these patent selections was then mined for accession numbers using the EPMC text-mining pipeline.\n\nThe performance assessment characteristics of the text-mining pipeline have been previously reported as 97.45% precision/59.6% recall for ENA and 94.63% precision/91.36% recall for PDB accession references when calibrated against an open access full-text corpus from EPMC14. No large-scale validation of the pipeline has been performed on the patent literature. However, manual inspection on a subset of 110 entries indicated that the approximate precision of the system was 99% and recall was 93%. Overall then the accuracy of the system appears to be higher when working with patents. This is possibly due to that fact that most citation-positive patents contain multiple exemplars whereas many research articles only include one. This would reduce the incidence of false negatives.\n\nThe EPMC metadata and text-mining results used in this study can be accessed or generated via Europe PMC’s RESTful API which gives access to search tools with citation-count sort order and data citation features. For example, to get all the PDB citations text-mined in the articles published in EPMC in 2014 go to http://www.ebi.ac.uk/europepmc/webservices/rest/search?query=PUB_YEAR:2014 and then for each of those get the accessions identifiers (e.g. for PMID 22517515 the query is http://www.ebi.ac.uk/europepmc/webservices/rest/MED/22517515/textMinedTerms/ACCESSION). The ENA accession data used here was obtained from EMBL release 124 (described in detail here ftp.ebi.ac.uk/pub/databases/ena/sequence/release/doc/relnotes.txt). The data are public and available at: ftp.ebi.ac.uk/pub/databases/embl/release/ or through the ENA Browser and REST API. We used the primary accession identifiers and deposition article PMIDs found in the flat XML files for each entry, and included all ENA data classes with the exception of the WGS (whole genome shotgun) depositions because these are lower level assemblies with sparse or no annotation information and so less likely to be cited in publications.\n\nThe PDB data was obtained from the 2 September 2015 release of the PDB. PDB has a weekly release cycle that is loaded and processed by the PDBe team. The PDBe database also contains information extracted from EPMC about additional PMID that reference or mention any given PDB accession identifier. This information is updated once a month. Citation data was extracted from the PDBe database and included information on PMID that mention the PDB identifier code or cite the primary citation describing the given PDB entry. Citation data is available via the PDBe API (See related publication call at http://www.ebi.ac.uk/pdbe/api/doc/) as well as on the individual PDBe entry pages (e.g. http://pdbe.org/3p8c and http://pdbe.org/3p8c/citations).\n\nEach record in a database has a unique accession number, a release or publication date, a series of revision dates, the bibliographic details of the deposition article, subsequent references associated with the generation of the data set and a list of references citing the source. By combining the metadata within the data resource with the citation information from EPMC we could identify the citation linked to the deposition article and hence distinguish between the initial citation event associated with the deposition article or the release of the data to the public, and track the secondary citations of a data entry (or annual cohorts of data entries) over time.\n\nThe data sets associated with the generation of Figure 1, Table 1, Table 2, Table 3 and Table 4, and Supplementary material Table 1 and Supplementary material Table 2 are provided (see Data availability). More specifically, data sets containing accession identifiers, deposition_PMID, deposition year, year of first_publication, and publication year of PMID were extracted from the data resources, and corresponding accession identifiers, citation year and number of data citations in that year were extracted from EPMC.\n\nThe merged data set contained the variables: [accession_id], [deposition_pmid], [deposition_year], [first_public_year], [pmid_publication_year], [citation_year], [citations]. For records that had a [pmid_publication_year] equal to a [citation_year], we reduced the corresponding [citations] count by one to remove the impact of the deposition citation. We then tabulated total citations for [first_public_year] (or [pmid_publication_year]) against accession/source article [citation_year].\n\nOur data analysis was carried out using the STATA 12 package (http://www.stata.com/products/).\n\n\nResults\n\nTo establish a baseline, we used citations of accession identifiers captured by the EPMC text-mining pipeline to provide a comprehensive picture of the annual data citation characteristics for ENA26, UniProt4, PDBe3, OMIM6, RefSNP, RefSeq5, Pfam27, InterPro28, Ensembl29, and ArrayExpress30.\n\nIn 2014, the ENA, PDB, and RefSNP accounted for 42.6%, 21.9% and 21.7% respectively of the total text-mined citations (Table 1). These proportions remained approximately constant throughout the sampled periods and hence provide a reference for comparison with the patent corpus below. In the Kafkas et al. study14 the corresponding percentages for a cohort of 486,472 articles published between 1990 and 2012 were 56.5%, 19.9% and 13.8%. We believe that the differences in these percentages can be attributed to the age structures of the two data corpuses, with the Kafkas set providing a more longitudinal view hence favouring well-established repositories such as ENA.\n\nAcc/Art is average accession references per article.\n\nUnsurprisingly, given the breadth of the biomedical literature, data citations of individual biomolecular resources are relatively infrequent in EPMC: for ENA, the proportion of citing papers in 2014 are 7,016/319,815, or 2.2%, and for PDB, 5,913/319,815 or 1.8%. Collectively the investigated databases are referenced in 6.5% of our EPMC sample (the EPMC search: “pub_year:2014 in_epmc:y”, conducted 20 Oct 2015, retrieved 319,815 articles).\n\nEstimates of secondary data citation in the scientific literature, whether measured via citation of an accession identifier in the article text or mentioned in the reference list (e.g. “1fho” or “doi: 0.2210/pdb1fho/pdb”) or via citation of the corresponding deposition article (e.g. “Blomberg et al.31”), need to make a distinction between citations that arise from the original act of data deposition and those that arise from the secondary citation of data. A further distinction, not investigated systematically here, could also be made according to whether the article citations come from one or more of the original author group – as above - or from an independent research group. The former practise would appear to be quite common for ENA depositions (D. Bousfield, unpublished observations). While this distinction seems straightforward in principle, different policies and deposition practices, as well as ambiguity of author names, make it difficult to distinguish these alternatives in a large-scale analysis. We note that the adoption of ORCID within publication workflows will support future disambiguation.\n\nCombining metadata stored in EPMC and the data resources allowed us to build up a picture, based on the summation of individual data elements, of how annual cohorts of accessions and deposition articles are cited over time (see Table 2). For example the PDB accession 2jhr that refers to the crystal structure of myosin-2 motor domain in complex with ADP-metavanadate and pentabromopseudilin was made public on 13 January 2009. The corresponding article for this deposition is PMID:19122661, entitled “The mechanism of pentabromopseudilin inhibition of myosin motor activity”, published later in 200932. At the time of our study, the deposition article had been cited a total of 9 times during the period 2009–2014 (the current list of citing articles can be found in EPMC using the query: cites:19122661_med). None of these papers cite the data accession identifier 2jhr. However, the database record was cited once by its accession identifier in PMID:21841195 (PMCID:3186370), “Structural basis for the allosteric interference of myosin function by reactive thiol region mutations G680A and G680V”. The actual statement from this paper provides a good example of how data citation occurs in narrative text: “This is very unusual, as the meta-vanadate is clearly visible in known wild-type myosin-2 structures that were obtained in the presence of ADP-VO3, like e.g. PDB IDs 2JJ9, 2JHR, and 2XO8”33. The text components recognised by the text-mining pipeline as being an accession citation of 2JHR are highlighted in bold. The text-mining pipeline also found 2JJ9 and 2XO8.\n\nThe rows show the year in which a PDB data entry was first made public. The columns denote the year in which a citation of that data accession was recorded. Thus each row displays the time-series of citations for the cohort of data entries published during a given year. Reasons why there are observations below the diagonal are discussed in the text. Mature cohorts (release years 2005–2011) were cited on average 0.21 times per accession per year.\n\nNote that whereas each data resource by definition contains references to the complete set of deposition articles, EPMC is incomplete in its full-text literature coverage and therefore will contain only a partial set of cited accession identifiers. In addition, the text-mining process will miss some citations (false negatives) and potentially create a small number of false positives (see Methods). These factors need to be kept in mind when interpreting the results shown in Table 2 and Table 3.\n\nSame format as per Table 3. Notice sustained levels of citation over time. Mature cohorts (publication year 2005–2011) were cited on average 6.73 times per source article per year.\n\nTable 2 displays the secondary citation of PDB accession identifiers, subject to above-mentioned caveats, published between 2005 and 2014. In theory, elements below the diagonal should all be zero as the non-zero numbers imply that the accession identifier has been cited before it has been made public. Some of these “below the diagonal” observations may be true false positives created by the text-mining process but also occur when the primary reference article in the database has been updated to a more recent publication. Non-zero “below diagonal” citations can also arise when authors embargo the publication of the data until after the publication of their own additional work citing the data set.\n\nTable 3 shows the corresponding picture for the continuing citation of the PDB deposition articles. Some similar “below diagonal” patterns were found and attributed to use of updated primary reference articles or occasionally genuine misalignments of the underlying archives.\n\nAs can be seen from Table 2 and Table 3 the citation of PDB data accession identifiers and PDB deposition articles remain high as the annual cohorts age. The average annual citation-rate for each deposition article in PDB is 6.7 and the annual average number of citations per accession identifier is 0.2. For ENA the corresponding statistics were 2.1 and 0.1 (Supplementary material Table 1 and Supplementary material Table 2 show the corresponding two data sets for ENA). In all four cases these citation rates are stable over time. It is worth noting that most ENA data depositions are not accompanied by a deposition article: 32,188,662 ENA entries in 2014 were not associated with a deposition article as compared to the 26,384,613 entries that were associated with 9,375 source articles. It is also worth noting that the text-mining of accession numbers in EPMC only occurs in the subset of the scientific literature where full-text is available in open access resources, hence these numbers represent a lower bound on direct data citations.\n\nPatents are frequently used as an indicator of broader societal value of research34–37. Importantly, it is estimated that only a small fraction of the scientific and technological innovation first reported in patents is subsequently disclosed in scientific literature sources38. During the creation of a patent it is essential to unambiguously identify the components of the invention and to provide extensive reviews of any prior art39. Thus we sought to address the question of how these requirements translate into data citation practices within patents.\n\nOur SureCHEMBL corpus of 188,589 full-text patents contained 7,923 patents with data citations (4.2% of the corpus). Data citations were most common in the description section – which usually constitutes by far the largest section of the document text. The breakdown by patent office shows that the majority of patents with data citations were from the US (see Supplementary material Table 3). The proportion of accessions found for the different repositories (Table 4) differed considerably from that of EPMC articles (see Table 1 for comparison) with RefSeq, ENA, RefSNP and UniProt dominating. The average number of cited accession identifiers per repository and per document (13.9) and the variance of these figures across the resources was also much higher than found for the full-text scientific literature corpus. Since the international patent code (IPC, see Methods) is a hierarchical patent classification system we can use its additional levels to probe the subject matter of accession-positive patents further. Figure 1 shows that patents with references to ENA and UniProt were extensively used to define biological entities in the IPC subclasses A61 (“Preparations for medical, dental or toilet purposes”), C07 (“Organic chemistry”) and C12 (“Microorganisms or enzymes”). The content profiles and scientific topics covered in the two corpuses – open access scientific publications and patent documents - are different and further work is needed to understand how this influences data citation rates.\n\nCompare the averages with those in Table 1. Acc/Pat is the average number of accessions per patent per repository.\n\nNote individual patents can have several IPC annotations – these percentages are based on summing all instances, i.e. “one code, one vote”. For example, 17% of the IPC codes annotating UniProt-positive patents were A61K. Key to coding: A61K preparations for medical, dental, or toilet purposes; C12N micro-organisms or enzymes; A61B diagnosis, surgery, intervention; C07K peptides; G01N investigating or analysing materials by determining their chemical or physical properties; C12P fermentation or enzyme-using processes to synthesise a desired chemical compound; C12Q measuring or testing processes involving enzymes or micro-organisms; C07D heterocyclic compounds. Note absence of A61B from the more biological data sets, compared to the presence of C07K.\n\n\nDiscussion\n\nCitation analysis is a cornerstone of research impact and evaluation and while the use and value of citation of research papers in the scholarly literature as a metric for research is much debated, the citation practices underpinning such analysis are generally unambiguous and well established. With research funders increasingly establishing open data policies, there is a requirement and interest in performance metrics that assess the reuse of open research data - whether to recognise and reward scientists, support the long-term management and sustainability of data archives or to understand the broader societal value derived from these policies. Quantitative analysis of data reuse, let alone estimating the value arising from this reuse, is challenging due to the diversity of data citation practices but also due to the many ways open research data can be used in further studies. As bioinformatics databases increasingly take on the role of dictionaries or “scientific instruments”40 we would expect that most of the use of biomolecular data resources (and consequently data reuse from these resources) is never cited, just as most literature searches, views or downloads from PubMed do not lead to a citation of the PubMed infrastructure.\n\nThis study set out to analyse data citation practices with the aim of describing secondary citation of data entries – as one indicator of data reuse - in full-text content available from EPMC (scientific papers) and SureCHEMBL (patents). We focused our efforts on the major biomolecular databases where high-quality curation processes and well-established links between literature and data resources allows us to dissect citations arising from data deposition articles from the secondary citations arising from reuse of this dataset in the scientific literature. Our approach can in principle be applied to all repositories by systematically bringing together metadata from the repository and from EPMC and is in itself a good illustration of the value that open access data and literature resources brings to the scientific community.\n\nThe need to separate deposition from reuse in quantitative studies of data citation has been noted previously41 but the complexity and manual analysis required often leads investigators towards aggregate analysis of a total citation rate. For instance, in an analysis of data citation practices across fields using the commercially available Thomson-Reuters Data Citation Index42 the average citation rate for data sets in many of the studied data resources was found to be close to one, suggesting that much of the ‘data citation’ found in this analysis was driven by data deposition publications. Separating out secondary citations by tracking them over time (Table 2 and Table 3) provides one, albeit limited, indicator of the reuse of the data sets in the scientific community. In the case of the two repositories we have analysed in detail, PDB and ENA, it demonstrates long-term reuse of data sets by the community.\n\nComparing the citation patterns arising from the deposition and reuse from ENA and PDB is instructive, as the mode of usage is very different for the two databases. While ENA is accessed directly by users on a daily basis, the more significant use is as a large reference repository that serves as the archival backend for user-focussed resources such as the genome browsers Ensembl and Ensembl genomes29. Most of the users that access the Ensembl resource on a daily basis are likely to be unaware of the relationship between ENA and Ensembl and hence would not cite the corresponding ENA entry.\n\nThe results in this study, taken together with previous work15,19,40,43 guards against reliance on metrics based on familiar approaches developed for the analysis of scientific papers. Such simplified citation metrics do not capture the many different forms of data reuse and heterogeneous and non-standard data citation practice in the biomedical literature. Data citation indexes also need to be developed that acknowledge that different patterns of use give different citation patterns for archival resources (e.g. PDB, ENA, GEO), reference knowledge bases (e.g. UniProt, Reactome, Human Protein Atlas), and secondary value-added resources (e.g. Interpro). Uniform quantitative indicators of data citation are inappropriate as they do not capture the usage patterns of the different resources.\n\nBiomolecular databases also exist within a network of mutual referencing and cross-mappings - just as literature articles build upon previous scholarly work and indicate this through citation there is a complex network of dependencies between bioinformatics databases - most of which is not visible in the primary literature44. Further work is needed to capture this usage pattern for assessments of the data journeys that occur through the extensive reuse and cross referencing of bioinformatics resources – and the corresponding return of investment from this scientific infrastructure.\n\nTo date investigations on data reuse have focused on the scientific literature. However, biological data resources are also extensively used by researchers in industry and in the second part of our study we started to address the use of bioinformatics databases in patents as a broader indicator of their industrial and societal value. Patent analyses have been extensively used to understand the industry and societal benefit from publicly funded research37,45–47 and full-text patents are available from several patent offices. The practice of large-scale text-mining of molecular entities from patents is well-established in chemistry48–51. However, to the best of our knowledge this is the first time that the usage and citation of bioinformatics data resources in the patent literature has been analysed; our beginning foray into this field demonstrates significant use of these resources to define biological concepts and subject matter in patent documents. Although the majority of data citation occurs in patent classes dealing with pharmaceutical and medical inventions (drugs, diagnostics and medical devices) the data also highlights a broad applicability of biomolecular resources in bio-based industries with usage in industrial biotechnology and consumer products, for example the definitions of enzymatic activity in washing powder.\n\n\nConclusion\n\nThe extensive and quantifiable reuse of data from biomolecular data resources demonstrates the critical role this infrastructure plays in life science research but also highlights the need for robust metrics of data use by the scientific community. Using the cross-referencing between literature repositories such as Europe PMC and the ENA and PDB archives we demonstrate how data citations arising from deposition of data in an archive can be distinguished from the subsequent reuse by the scientific community – an important distinction in research evaluation as the former provides an estimate of adoption of community best practice and/or compliance with open access guidelines, whereas the second is an indicator of the value created by these practices and guidelines. The study also demonstrates that measures based on literature citation may be more or less informative according to mode of use of a repository: large biological archives serve as foundations for other value-added resources. Individual data items from large repositories such as ENA may not be directly cited in the scientific literature but collectively forms important reference collections for e.g. pathogen detection or biodiversity research. Further work is needed to develop methods that classify and account for this mode of use, e.g. by quantifying database cross-linking via literature citation networks and identifier mapping.\n\nBy extending the analysis to patent documents we show that the biological data resources provide unambiguous definitions of biological entities for use in official documents such as patents. This shows that life science data resources transcend basic research and form a fundamental component of the digital knowledge management framework needed in a modern society. Hence, assessment of the use and value of scientific data repositories should include data from research articles, patents and perhaps other documents of record such as clinical guidelines, standards, and grant applications. Understanding how to establish robust indicators of data citation in these types of documents in addition to research articles remains an important challenge for further studies. The ecosystem of open literature and data resources can only be sustained if the creation of scientific and societal value can be properly assessed and the scientific and scholarly community needs to make a concerted effort to better cite data. Similar principles can be applied to other resources such as reagents and software. Finally we note that the insights from reviewing data citation patterns could be used to improve article level metrics, this is also an area of further investigation.\n\n\nData availability\n\nF1000Research: Dataset 1. Raw data for ‘Patterns of database citation in articles and patents indicate long-term scientific and industry value of biological data resources’, Bousfield et al., 2016. 10.5256/f1000research.7911.d11328152", "appendix": "Author contributions\n\n\n\nDB, JM and NB conceived of the study; DB, GP, JHK, SV, FG, VV, BA acquired the data; DB, GP, JHK, FG, AB, GC, SV, JM and NB analysed the data and wrote the manuscript.\n\n\nCompeting interests\n\n\n\nNone of the authors declare competing interests.\n\n\nGrant information\n\nSupport for some of the analysis described here (FG) was provided by EC H2020 Grant 643410, OpenAIREH2020. GP would like to acknowledge the Wellcome Trust Strategic Awards [WT086151/Z/08/Z, WT104104/Z/14/Z] for the development of SureChEMBL.\n\nWe confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe thank all our colleagues who diligently deposit data together with high quality metadata annotations in public biomolecular resources.\n\n\nSupplementary Material\n\nSupplementary tables for 'Patterns of database citation in articles and patents indicate long-term scientific and industry value of biological data resources'.\n\nClick here to access the data.\n\n\nReferences\n\nBaxevanis AD, Bateman A: The Importance of Biological Databases in Biological Discovery. Curr Protoc Bioinformatics. 2015; 50: 1.1.1–1.8. PubMed Abstract \| Publisher Full Text\n\nNakamura Y, Cochrane G, Karsch-Mizrachi I, et al.: The International Nucleotide Sequence Database Collaboration. Nucleic Acids Res. 2013; 41(Database issue): D21–D24. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGutmanas A, Alhroub Y, Battle GM, et al.: PDBe: Protein Data Bank in Europe. Nucleic Acids Res. 2014; 42(Database issue): D285–D291. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUniProt Consortium: UniProt: a hub for protein information. Nucleic Acids Res. 2015; 43(Database issue): D204–D212. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPruitt KD, Brown GR, Hiatt SM, et al.: RefSeq: an update on mammalian reference sequences. Nucleic Acids Res. 2014; 42(Database issue): D756–D763. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOnline Mendelian Inheritance in Man, OMIM®. 2016. Reference Source\n\nNucleic Acids Research Database Summary. 2015. Reference Source\n\nBoulton R: Science as an open enterprise. London: Royal Society. 2012; 104. Reference Source\n\nRebholz-Schuhmann D, Grabmüller C, Kavaliauskas S, et al.: A case study: semantic integration of gene-disease associations for type 2 diabetes mellitus from literature and biomedical data resources. Drug Discov Today. 2014; 19(7): 882–9. PubMed Abstract \| Publisher Full Text\n\nSchurer SC, Vempati U, Smith R, et al.: BioAssay ontology annotations facilitate cross-analysis of diverse high-throughput screening data sets. J Biomol Screen. 2011; 16(4): 415–426. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGaulton A, Overington JP: Role of open chemical data in aiding drug discovery and design. Future Med Chem. 2010; 2(6): 903–907. PubMed Abstract \| Publisher Full Text\n\nSchwede T: Protein modeling: what happened to the “protein structure gap”? Structure. 2013; 21(9): 1531–1540. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRung J, Brazma A: Reuse of public genome-wide gene expression data. Nat Rev Genet. 2013; 14(2): 89–99. PubMed Abstract \| Publisher Full Text\n\nKafkas Ş, Kim JH, McEntyre JR: Database citation in full text biomedical articles. PLoS One. 2013; 8(5): e63184. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPiwowar HA, Carlson JD, Vision TJ: Beginning to track 1000 datasets from public repositories into the published literature. Proc Am Soc Info Sci Tech. 2011; 48(1): 1–4. Publisher Full Text\n\nBarrett T, Wilhite SE, Ledoux P, et al.: NCBI GEO: archive for functional genomics data sets--update. Nucleic Acids Res. 2013; 41(Database issue): D991–D995. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPANGAEA: Data Publisher for Earth & Environmental Science. Publisher Full Text\n\nSanderson MJ, Donoghue MJ, Piel WH, et al.: TreeBASE: a prototype database of phylogenetic analyses and an interactive tool for browsing the phylogeny of life. Am J Bot. 1994; 81(6): 183. Reference Source\n\nBelter CW: Measuring the value of research data: a citation analysis of oceanographic data sets. Browman HI, ed. PLoS One. 2014; 9(3): e92590. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKafkas Ş, Kim JH, Pi X, et al.: Database citation in supplementary data linked to Europe PubMed Central full text biomedical articles. J Biomed Semantics. 2015; 6(1): 1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPapadatos G, Davies M, Dedman N, et al.: SureChEMBL: a large-scale, chemically annotated patent document database. Nucleic Acids Res. 2016; 44(D1): D1220–D1228. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEurope PMC Consortium: Europe PMC: a full-text literature database for the life sciences and platform for innovation. Nucleic Acids Res. 2015; 43(Database issue): D1042–D1048. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRebholz-Schuhmann D, Arregui M, Gaudan S, et al.: Text processing through Web services: calling Whatizit. Bioinformatics. 2008; 24(2): 296–298. PubMed Abstract \| Publisher Full Text\n\nEisinger D, Tsatsaronis G, Bundschus M, et al.: Automated Patent Categorization and Guided Patent Search using IPC as Inspired by MeSH and PubMed. J Biomed Semantics. 2013; 4(Suppl 1): S3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThe Espacenet patent family. 2015. Reference Source\n\nSilvester N, Alako B, Amid C, et al.: Content discovery and retrieval services at the European Nucleotide Archive. Nucleic Acids Res. 2015; 43(Database issue): D23–D29. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFinn RD, Bateman A, Clements J, et al.: Pfam: the protein families database. Nucleic Acids Res. 2014; 42(Database issue): D222–D230. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHunter S, Jones P, Mitchell A, et al.: InterPro in 2011: new developments in the family and domain prediction database. Nucleic Acids Res. 2012; 40(Database issue): D306–D312. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFlicek P, Amode MR, Barrell D, et al.: Ensembl 2014. Nucleic Acids Res. 2014; 42(Database issue): D749–D755. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPetryszak R, Burdett T, Fiorelli B, et al.: Expression Atlas update--a database of gene and transcript expression from microarray- and sequencing-based functional genomics experiments. Nucleic Acids Res. 2013; 42(Database issue): D926–D932. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlomberg N, Baraldi E, Sattler M, et al.: Structure of a PH domain from the C. elegans muscle protein UNC-89 suggests a novel function. Structure. 2000; 8(10): 1079–1087. PubMed Abstract \| Publisher Full Text\n\nFedorov R, Böhl M, Tsiavaliaris G, et al.: The mechanism of pentabromopseudilin inhibition of myosin motor activity. Nat Struct Mol Biol. 2009; 16(1): 80–88. PubMed Abstract \| Publisher Full Text\n\nPreller M, Bauer S, Adamek N, et al.: Structural basis for the allosteric interference of myosin function by reactive thiol region mutations G680A and G680V. J Biol Chem. 2011; 286(40): 35051–35060. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBessen J: The value of U.S. patents by owner and patent characteristics. Res Policy. 2008; 37(5): 932–945. Publisher Full Text\n\nNarin F, Hamilton KS, Olivastro D: The increasing linkage between U.S. technology and public science. Res Policy. 1997; 26(3): 317–330. Publisher Full Text\n\nMinguillo D, Thelwall M: Which are the best innovation support infrastructures for universities? Evidence from R&D output and commercial activities. Scientometrics. 2015; 102(1): 1057–1081. Publisher Full Text\n\nBenson CL, Magee CL: Quantitative determination of technological improvement from patent data. Huy NT, ed. PLoS One. 2015; 10(4): e0121635. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBregonje M: Patents: A unique source for scientific technical information in chemistry related industry? World Patent Information. 2005; 27(4): 309–315. Publisher Full Text\n\nGrubb PW, Thomsen PR: Patents for Chemicals, Pharmaceuticals, and Biotechnology. Oxford University Press; 2010. Reference Source\n\nHine C: Databases as Scientific Instruments and Their Role in the Ordering of Scientific Work. Soc Stud Sci. 2006; 36(2): 269–298. Publisher Full Text\n\nPiwowar HA: Foundational Studies for Measuring the Impact, Prevalence, and Patterns of Publicly Sharing Biomedical Research Data. 2010. Reference Source\n\nRobinson-García N, Jiménez-Contreras E, Torres-Salinas D: Analyzing data citation practices using the Data Citation Index. arXiv. cs.DL:n/a-n/a. 2015. Publisher Full Text\n\nVision TJ, Piwowar HA: Data reuse and scholarly reward: understanding practice and building infrastructure. 2013. Publisher Full Text\n\nCook CE, Bergman MT, Finn RD, et al.: The European Bioinformatics Institute in 2016: Data growth and integration. Nucleic Acids Res. 2016; 44(D1): D20–D26. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchankerman M, Pakes A: Estimates of the Value of Patent Rights in European Countries During the Post-1950 Period. Cambridge, MA: National Bureau of Economic Research; 1985. Publisher Full Text\n\nBacchiocchi E, Montobbio F: Knowledge diffusion from university and public research. A comparison between US, Japan and Europe using patent citations. J Technol Transf. 2009; 34(2): 169–181. Publisher Full Text\n\nPackalen M, Bhattacharya J: Words in Patents: Research Inputs and the Value of Innovativeness in Invention. Cambridge, MA: National Bureau of Economic Research; 2012. Publisher Full Text\n\nJessop DM, Adams SE, Murray-Rust P: Mining chemical information from open patents. J Cheminform. 2011; 3(1): 40. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAkhondi SA, Klenner AG, Tyrchan C, et al.: Annotated chemical patent corpus: a gold standard for text mining. PLoS One. 2014; 9(9): e107477. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHersey A, Chambers J, Bellis L, et al.: Chemical databases: curation or integration by user-defined equivalence? Drug Discov Today Technol. 2015; 14: 17–24. PubMed Abstract \| Publisher Full Text\n\nSenger S, Bartek L, Papadatos G, et al.: Managing expectations: assessment of chemistry databases generated by automated extraction of chemical structures from patents. J Cheminform. 2015; 7(1): 49. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBousfield D, McEntyre J, et al.: Dataset 1 in: Patterns of database citation in articles and patents indicate long-term scientific and industry value of biological data resources. F1000Research. 2016. Data Source" }	[ { "id": "12391", "date": "22 Feb 2016", "name": "Mark Parsons", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 advances our understanding of how data are used and referenced. It is well-written, well-referenced, and the methods are appropriate. The data are explained and seem to be available and usable. The conclusions are reasoned and sound. The paper should be approved, and I suggest some minor improvements that would help clarify the methods and make the document more relevant and useful to a broader audience.The paper analyzes the data citation landscape in the life sciences, specifically around key databases (ENA, PDB) and the open access journals in EPMC and the corpus of patent documents in SureChEMBL. The study findings, however, have implications well beyond life sciences. As such, the paper would benefit from more explanatory context. Some of this explanation was in the discussion section, but it would be helpful to the general reader if more of this was included in the introduction. Also I consider myself expert in issues of data citation, but my work has largely been in the geosciences, which has a different data and literature publishing workflow. I was a little confused by the distinction between deposition citations and reuse citations. I think I had it figured out by the end of the paper, but it would have helped to have a clear explanation, early in the article, of the typical biomedical data and article publication process and how that defines a distinction between deposition citations and reuse citations. I was also not fully clear on how deposition and secondary citations were distinguished in the methodology.Similarly, a bit more detail on the methodology would be useful. For example, the references explain the text-mining techniques, but another paragraph or two in this article that summarized the approach would help the reader understand the approach.Finally, while I found the discussion and conclusion sections strong, I was thrown by the last sentence in the first paragraph of the discussion. Is this to imply that data citation decreases as a database becomes more established? (I do not have access to the cited article). If that is what is meant, there should be more discussion on the implications for further citation analysis work of this type.All in all, a useful and important paper. I would be very intrigued to see the methodology applied to other disciplines.", "responses": [] }, { "id": "12390", "date": "31 Mar 2016", "name": "Timothy W. Clark", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 look at citation patterns and frequencies of some important and representative bioinformatics data resources (ENA, ePDB) in the professional and patent literature, based on textmining for accesssion numbers in the Euro PubMed Central Open Access corpus, and patent documents in SureChEMBL.The authors provide resources to reproduce their results using RESTFUL APIs at EMBL. As might be expected of resource providers interested to know how the services they provide are taken up - the authors deal exclusively with “citation for reuse”. By this is meant citation of database entry accession numbers to indicate they were used as a source of further analyses, rather than to mark them as an original primary data deposition. Analysis of citations for reuse documents the value of archived data for use beyond the study that produced it.The methodology is well described. Authors note that “citation analysis is a cornerstone of research impact and evaluation”. Their analysis demonstrates distinct usage patterns for deposition and reuse in ENA and ePDB and for patents vs. articles as citing documents.By quantifying the extent of reuse of data from EMBL bimolecular resources the authors demonstrate the importance in biomedical research of such resources .This is a well written paper with a carefully designed methodology which quantifies the role of key EMBL data rescues in the biomedical and bio-industrial community.", "responses": [] }, { "id": "12982", "date": "13 Apr 2016", "name": "Ben Johnson", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThose involved in thinking about the scientific and societal impact of research will know that the complexities in data sharing, citation and reuse practices often hinder us from developing a quantitative understanding of the value of data. This has implications for how data are treated as an important output of research across a wide variety of fields, including in institutional and funder assessment frameworks.The line of inquiry taken in this study to separate original citation from later reuse provides us with a one very promising way forward for how we might start to address some of these complexities. In this way, the study makes a valuable contribution to our understanding of how the impact of scientific data may usefully be quantified and tracked in future.In addition, the study shows us the importance of open and carefully-maintained data infrastructure -- not just for data but also for scientific literature and patents - if we are to understand the important role that data plays in a modern, knowledge-based economy. The importance of high quality data sources is clear; the scope for others to extend this methodology to other disciplines and fields may depend on similar high quality sources and infrastructure being in place. If further work is needed to improve the data infrastructure then this study in part provides a good rationale for this.This is a very well-designed study with a clear methodology. The paper is very well-written and the discussion section in particular provides a valuable and thought-provoking contribution to the debates.", "responses": [] } ]	1	https://f1000research.com/articles/5-160
https://f1000research.com/articles/5-158/v1	11 Feb 16	{ "type": "Method Article", "title": "Prediction of fine-tuned promoter activity from DNA sequence", "authors": [ "Geoffrey Siwo", "Andrew Rider", "Asako Tan", "Richard Pinapati", "Scott Emrich", "Nitesh Chawla", "Michael Ferdig", "Andrew Rider", "Asako Tan", "Richard Pinapati", "Scott Emrich", "Nitesh Chawla", "Michael Ferdig" ], "abstract": "The quantitative prediction of transcriptional activity of genes using promoter sequence is fundamental to the engineering of biological systems for industrial purposes and understanding the natural variation in gene expression. To catalyze the development of new algorithms for this purpose, the Dialogue on Reverse Engineering Assessment and Methods (DREAM) organized a community challenge seeking predictive models of promoter activity given normalized promoter activity data for 90 ribosomal protein promoters driving expression of a fluorescent reporter gene. By developing an unbiased modeling approach that performs an iterative search for predictive DNA sequence features using the frequencies of various k-mers, inferred DNA mechanical properties and spatial positions of promoter sequences, we achieved the best performer status in this challenge. The specific predictive features used in the model included the frequency of the nucleotide G, the length of polymeric tracts of T and TA, the frequencies of 6 distinct trinucleotides and 12 tetranucleotides, and the predicted protein deformability of the DNA sequence. Our method accurately predicted the activity of 20 natural variants of ribosomal protein promoters (Spearman correlation r = 0.73) as compared to 33 laboratory-mutated variants of the promoters (r = 0.57) in a test set that was hidden from participants. Notably, our model differed substantially from the rest in 2 main ways: i) it did not explicitly utilize transcription factor binding information implying that subtle DNA sequence features are highly associated with gene expression, and ii) it was entirely based on features extracted exclusively from the 100 bp region upstream from the translational start site demonstrating that this region encodes much of the overall promoter activity. The findings from this study have important implications for the engineering of predictable gene expression systems and the evolution of gene expression in naturally occurring biological systems.", "keywords": [ "Promoter activity", "Gene expression", "Expression prediction", "DREAM challenges", "Machine learning", "Gene regulation", "DNA sequence", "Transcription modeling" ], "content": "Introduction\n\nTranscription is a fundamental step in the decoding of information encoded in DNA into phenotypes. Therefore, knowledge of transcriptional regulation is crucial for understanding the natural variation of gene expression1–5 and for the accurate engineering of predictable gene expression systems6–8. While transcriptional regulation is one of the most highly studied areas in biology, the ability to quantitatively predict gene expression from DNA sequence remains inadequate9,10. Knowledge of transcription factors and their cognate binding sites continues to grow and has enhanced our ability to make qualitative predictions about gene expression. For example, a number of transcription factors are now well known to be involved in differentiation of stem cells into specific cell types, leading to potentially clinically useful applications such as induced pluripotent stem cells11. Inspite of this progress, only limited quantitative predictions of gene expression are possible6–8,12,13. Knowledge that promoter sequences of genes encode both qualitative (e.g. when to switch a gene on and off) and quantitative properties (e.g. precise levels and noise) of gene expression is implied by the heritable nature of these attributes1–3,14. It is becoming increasingly clear that while transcription factors are critical in gene regulation, regulatory outputs are ultimately determined by co-operation between regulators in complex circuits15–17 and with chromatin states18–21. In particular, transcription factors compete for DNA binding sites with nucleosomes22,23. The information for nucleosome binding is largely encoded in the DNA sequence24–27, even though in vivo nucleosome occupancy is highly dynamic25,28,29. Quantitative models of gene expression, therefore, benefit from the integration of nucleosome and transcription factor binding data10,23,30.\n\nA key barrier to quantitative modeling of gene expression using promoter sequence has been the lack of experimental methods for accurately measuring transcript levels. DNA microarrays and RNA-seq are the most widely-used systems for measuring transcript abundance, but this measurement can reflect many effects including promoter sequence, genomic position of a gene and post-transcriptional regulation of mRNA levels by processes like mRNA degradation. In addition, microarray and RNA-seq can be affected by systematic biases arising from sequence dependent hybridization kinetics31 and sequence dependent read-depth coverage32, respectively. To overcome these limitations, approaches based on promoters fused to fluorescent reporters have been developed to generate direct, real-time measurement of promoter activity with high accuracy33. This has been applied in large libraries of synthetic bacterial promoters thereby generating new insights on combinatorial cis-regulation8. It was not until recently that the first large-scale library of naturally occurring promoters of any eukaryote fused to yellow fluorescent protein (YFP) became available30. 110 yeast ribosomal protein (RP) promoters were fused to YFP and integrated into a different strain at a fixed genomic location, hence alleviating both post-translational and genomic context related effects30. Consequently, this data set is very well poised for the computational modeling of the relationship between promoter sequence and transcription activity of a eukaryotic promoter.\n\nTo provide a fair assessment of the relationship between promoter sequence and quantitative transcript levels, the Dialogue for Reverse Engineering Assessments and Methods (DREAM) organized an open community challenge in 2011 (details of the challenge as well as an overview of participating teams is provided in reference 34), inviting participants to address this question using promoter activities of the RP promoter library that was not yet published30. Participants were provided with the activities of 90 promoters and their corresponding promoter sequences and challenged to predict the activity of 53 promoters whose activities were known only to the organizers of the challenge (Figure 1A). After a period of three months, the challenge organizers independently assessed the performance of models from 21 teams using four different statistical tests. Our team, Fighting Irish Systems Team (FIrST), attained the best performance status on the basis of a combined score by the DREAM consortium in predicting the activities of these 53 promoters (Spearman correlation between predicted and actual activities r = 0.65, P = 0.002). Our approach was built upon three key propositions: i) transcription factor binding and nucleosome binding, as well as other regulatory signals are encoded in DNA9,10,12,27, ii) if i) is true, then explicit prior knowledge of transcription factor and nucleosome binding is not a mandatory prerequisite for prediction of promoter activity if training data is available. That is, an unbiased approach that explores the associations between DNA sequence patterns and promoter activity should be able to rediscover patterns that relate to the observed activity. To do this, we used machine learning methods to iteratively explore the association between promoter activity and DNA sequence patterns in 100 bp windows of promoter sequence. We considered sequence patterns such as k-mers (k = 1 to k = 5), homopolymer stretches, nucleosome binding and three mechanical properties of DNA (bendability35, deformability36 and stiffness37). Based on iterative exploration of different machine learning models, we established that a support vector machine (SVM) was the most predictive of promoter activity based on specific sequence patterns in the 100 bp upstream of the translation start site (TrSS). Our model outperformed those which applied transcription factor binding sites of known RP promoters34, implying that other sequence patterns besides transcription factor binding sites can help in fine-tuning gene expression. Indeed, among the predictive features employed by our model were poly(dT-dA) tracts that occlude nucleosomes; these have since been applied to fine-tune gene expression beyond resolutions attainable by transcription factor site mutations38. Our study expands the understanding of sequence patterns that could potentially be useful in engineering fine-tuned gene expression.\n\n(A) Training data consisted of DNA sequences for 90 yeast RP promoters whose activities were experimentally determined30,34. DNA sequences for blinded test set of 53 promoters whose activity was hidden also experimentally determined but withheld from the challenge participants was also provided. (B) Outline for strategy of modeling promoter activity. Each promoter was segmented into 100 bp non-overlapping windows with the full promoter regarded as a separate window. For each window, DNA sequence features were extracted and feature selection using a linear regression wrapper performed prior to machine learning. Performance of machine learning models trained on each window was determined in 5- and 10-fold cross-validations using Pearson correlation.\n\n\nMethods\n\nThe training data composed of DNA sequence for 90 yeast RP promoters with known activities and a test data set of 53 promoters was downloaded from the DREAM challenge website (https://www.synapse.org//#!Synapse:syn2820426/wiki/71012). Details of promoter construction are available from Zeevi et al. 201130 and the DREAM website. Briefly, the organizers considered the promoter region as the sequence 1200 bp upstream of a gene or until the nearest gene. Each promoter was linked to a URA3 selection marker and inserted into the same fixed genomic location of a master yeast strain containing the YFP gene. In total, 110 natural RP promoter strains and 33 strains with synthetically mutated RP promoters were constructed. As a control for experimental variation, all these strains contained a control promoter (TEF2) driving the expression of red fluorescent protein (mCherry). The mCherry, TEF2, URA3, RP promoter and YFP were all a single contiguous DNA sequence arranged in that order. Measurements of the mCherry expression levels and replicates of promoters had very low variation, enabling the distinction between any two promoters with activities differing by as little as ~ 8%. The promoter activity was determined as the amount of YFP fluorescence produced during the exponential growth phase, divided by the integral of the OD during the same period. The promoter activity measures the average amount of YFP produced from each promoter, per cell, per second during the exponential phase.\n\nEach promoter sequence was divided into 100 bp non-overlapping windows. The full promoter sequence was considered as another window. To extract information from each of the windows, we considered the frequencies of specific sequences in k-mers (k = 1 to 5), length of homopolymeric stretches DNA, mechanical properties (deformability, bendability and stiffness) and nucleosome binding. K-mer counts were performed using custom scripts. DNA mechanical properties were computed using workflows constructed in the Taverna Workbench version 2.2.053 and BioMoby web-services (accessed in August 2011) imported from the Molecular Modeling and Bioinformatics Group, Barcelona, Spain54. Bendability was estimated based on trinucleotide parameters obtained from DNase I digestion and nucleosome binding data35. Deformability was based on parameters from the analysis of protein-DNA crystallography structures36. Bending stiffness was based on bending free energy using the near-neighbor model37. Nucleosome binding was based on trinucleotide preferences55.\n\nFor each window, feature selection was performed using a linear regression wrapper in the WEKA machine learning toolkit version 3.456 to select feature combinations that are most predictive of promoter activity. Performance of feature combinations was tested using 5- and 10-fold cross validation.\n\nThree models implemented in the WEKA toolkit56 were considered: SVM regression using sequential minimal optimization (SMO), linear regression and regression trees. Models were trained using 66% of the data and tested using 34%, and included only the features that were selected as important by the linear regression wrapper. Performance was determined using Pearson correlation between model predictions and actual promoter activities computed in R version 2.11.1. The SVM model was selected for refinement based on high performance compared to the other models.\n\nPromoter activities were not available to the participants of the challenge. We applied the ensemble of 501 SVMs built from 500 different training/test sets in which 80% of the data was used in training and 20% in testing and a single SVM validated by 66% training set and 34% testing sets. Each SVM model utilized the 24 features selected by a linear regression wrapper as most predictive of promoter activity. To predict activities of the DREAM6 test set, the 24 features were extracted from the upstream 100 bp sequence for each promoter. Predictions were then made using each of the SVM models and averaged to obtain the final predictions.\n\nPredictions from the SVM ensemble were submitted through the DREAM website to the organizers for a blinded evaluation on the test set. The DREAM organizers used four statistics and corresponding P-values to evaluate the performance on the test set34. Details of the equations used for these statistics have been published separately by the DREAM6 Promoter Prediction Consortium34.\n\n1. Pearson correlation between predicted and observed activities for each model submitted: To generate a P-value for observing a Pearson correlation coefficient of the same magnitude or smaller than that of a given participant, a null distribution was generated by randomly sampling predictions from other teams and repeating this 10,000 times34.\n\n2. Spearman correlation for participant between ranks of the predicted and actual ranks of promoter activities: A P-value was then generated using a null distribution obtained from randomly sampling the predictions made by the other participants. The process was repeated 10,000 times34.\n\n3. Chi-square distance metric measuring the distance between predicted and actual promoter activities: To generate a P-value for observing a chi-square distance metric of the same magnitude or smaller than that of a given model submission, a null distribution was generated by randomly sampling predictions from other teams and repeating this 10,000 times34.\n\n4. A rank distance metric measuring the difference in ranks between predicted ranks and actual ranks of promoter activities. A P-value was generated from a null distribution obtained by randomly sampling predicted ranks from other teams, repeating this 10,000 times.\n\nThe overall score was defined as the product of the four P-values34. All these scores were computed using R version 2.11.1.\n\n\nResults\n\nThe challenge organizers provided DNA sequences and promoter activities - the average rate of YFP production from each promoter, per cell per second, during the exponential phase - for 90 RP promoters (training set) and another set of 53 promoters whose activity was withheld from participants (test set)30. We first partitioned the promoter sequences into 100 bp non-overlapping windows, extracted specific DNA features from each window and considered the full promoter sequence as its own window (Figure 1B). The features considered were k-mers (k = 1 to 5), length of homopolymeric stretches, nucleosome positioning and DNA mechanical properties (bendability, deformability and stiffness). For each window, we performed feature selection using a linear regression wrapper, then explored three different machine learning methods (SVM, linear regression and regression trees) to learn the association between features in the window and promoter activity (Figure 1B). The performance in each window was assessed by Pearson correlation using 5- and 10-fold cross-validations on the training data. We observed very poor correlation (r « 0.5) between predicted and actual promoter activities except when using the window comprising 100 bp from the TrSS. Therefore, we focused the SVM model on this window using 23 features (Table 1) selected by the linear regression wrapper. A test of this model on 1000 randomized splits of the data (66% training and 34% testing sets) gave an average Pearson correlation of 0.78. The performance of machine learning models can be biased by the training/test data set used. Therefore, to reduce this bias, we obtained an additional 500 SVM models trained on randomly sampled sets of 80% of the data and validated on the remaining 20%. In the DREAM test set (activities for this set were withheld from participants), we used the SVM models to make predictions for each promoter. For each promoter, the predicted activity was the average of predictions across all the ensemble of SVMs based only on the 100 bp upstream of the TrSS. These predicted activities were then submitted to the DREAM consortium for evaluation34.\n\nA total of 21 teams participated in the challenge (https://www.synapse.org//#!Synapse:syn2820426/wiki/71013). Predictions from our team had a Spearman correlation of 0.65 (P = 0.002, Figure 2A) to the actual activities, Pearson correlation of 0.65 (P = 0.003), chi-squared (χ2) distance metric of 52.62 (P = 0.508) and R2 statistic measuring the difference in ranks between predicted and actual promoter activities of 35.85 (P = 0.004). The P-values were generated from the probability of obtaining a comparable or lower performance using a null distribution in which predictions were made by randomly choosing an activity for each promoter amongst all the 21 participating teams. A combined score based on the negative logarithm (base 10) of the geometric mean of the P-values for all the 4 scores ranked our team first34 (Figure 2B), with significant P-value in three out of four of the statistical tests used for evaluation. Further, although we were not ranked first in the χ2 distance metric, our model performed the most consistently across the multiple assessment metrics, suggesting a robustness of the method. A detailed comparison of the teams was published previously by the DREAM consortium34.\n\n(A) Correlation between predicted activity by the SVM model and actual promoter activity of 53 promoters whose activity was not available to participants. (B) Performance of team FIrST relative to other 20 teams based on a combined score.\n\nThe final SVM models utilized only 23 features consisting of the frequencies of the mononucleotide G, dinucleotide GT, 6 different trinucleotides, 12 different tetranucleotides, length of poly(dT) and poly(dA-dT) tracts (Table 1). The relative importance of these features based on weights for the SVM models is provided (see Data availability). The feature with the highest weight was the frequency of the mononucleotide G, correlating negatively with promoter activity. For many of these features there was no clear link to underlying mechanisms of gene regulation. However, it is possible that some of the k-mers may be implicitly linked to transcription factor binding sites. That is, the combination of different k-mer features could capture the binding motifs of specific transcription factors. For example the second most important feature in the SVM was the tetranucleotide ACCC which also occurs in the Rap1 binding site motif39. In addition, frequencies of different k-mers could impact the DNA mechanical structure40. Among the features identified by the SVM model were poly(dT) and poly(dT-dA) tracts which influence the rigidity of DNA24,26, thereby directly impacting nucleosome binding. Furthermore, insertion of poly(dT-dA) sequences into promoters can be used to regulate gene expression to a finer degree and at more gradual intervals than could be attained by transcription factor binding site mutations38. Some transcription factors are also highly dependent on the ability of DNA to bend41–43. In particular, TATA binding protein (TBP), which binds to the TATA box, is important for regulating the activity of RP promoters42,44,45. Another directly biologically relevant feature identified by the SVM was the deformability of DNA36,46. Promoters of low activity had more deformable DNA than those of high activity (Figure 3, P = 0.008). This was particularly evident at 40 to 60 bp from the TrSS when comparing the top 20 promoters with the highest versus those with the lowest activity (Figure 3).\n\nAmong the top 20 promoters with extreme activities (high and low), significant deviation in deformability occurs at the -40 to -60 bp region from the TrSS (T-test P = 0.008).\n\nFinally, some of the features may affect mRNA stability, especially given their potential location downstream of the transcription start sites (TSS). Besides sequence features in the 5’UTR that are close to the TSS could affect transcription, translation and mRNA stability.\n\nUnderstanding the biases in prediction accuracy could provide biological insights into promoter classes and allow for refinement of models. Therefore, we investigated relationships between the nature of the test promoters and the magnitude of prediction error made by our model. Among the 53 test promoters provided by the DREAM challenge, 20 were natural yeast RP promoters while 33 were variants of these promoters with specific synthetic mutations introduced. These mutations included changes in the binding sites of the TBP, Rap1, Fhl and Sfp1, as well as introduction of nucleosome disfavoring sequences and random mutations. At the time of the challenge, participants were not aware of these mutations. The performance of our model on the set of natural promoters was much higher (Pearson correlation r= 0.73, P = 0.0003) compared to that for the mutated promoters (Pearson correlation r= 0.57, P = 0.0005). The prediction error was significantly less for natural promoters versus the mutated promoters (Student’s t-test, P = 0.01, Figure 4A). This could partly be due to the composition of the training set, which contained only natural promoters. Similar poor performance was also observed in the models obtained from other teams34. In addition, most of the synthetic mutations were introduced at promoter locations residing outside of the 100 bp region from the TrSS and could not therefore be detected by our model. We also examined the correlation between the observed promoter activity and the prediction error. Promoters of low activity had larger prediction error (Pearson correlation between promoter activity and prediction error r= -0.31, P = 0.02, Figure 4B). Notably, natural promoters had slightly lower activity compared to synthetic promoters (P = 0.02) so the correlation between activity and prediction error may be a consequence of the low predictability of synthetic promoters. Thus, future models may benefit from data on activities of mutated promoters, which could enable a more accurate modeling of the impact of mutation on specific transcription factor binding sites.\n\n(A) Natural promoters had a lower prediction error compared to synthetically mutated promoters. (B) Prediction error is negatively correlated to promoter activity.\n\n\nDiscussion\n\nThe quantitative modeling of gene expression has the potential to enhance our understanding of how gene regulation is fine-tuned in natural populations and has implications for the design of predictable gene expression systems. The DREAM6 challenge data set for promoter activity prediction was a unique opportunity to evaluate the predictability of gene expression from its promoter sequence. Given that all promoters were derived from natural yeast RP promoters that are expressed in the exponential phase30, the challenge posed was more targeted towards DNA sequence patterns that fine-tune gene expression rather than simply determine the ‘on/off’ expression status. RP transcription regulation occurs in a highly coordinated manner and is critical for growth, allowing cells to adjust their protein synthesis capacity to physiological needs47,48. This is especially crucial as RP gene expression accounts for 50% of transcripts produced by RNA polymerase II49 and their dysregulation leads to reduced fitness47,48. The yeast genome contains 137 RP genes, of which 19 encode a unique RP and 59 are duplicated. The proper functioning of ribosomes requires that all the ribosome components be expressed in equimolar concentrations50 while simultaneously remaining responsive to physiological needs51,52. This is potentially challenging given the copy-number differences between the RP genes because high copy number genes generally show increased expression. The regulatory mechanisms underlying this fine-tuned regulation are not known. By accurately predicting the activity of the RP genes using the promoter sequences, we demonstrate that a considerable amount of this information is encoded in the DNA sequence.\n\nIt is intriguing that our model did not explicitly use transcription factor binding site information and focused only on the 100 bp upstream region. Some of the features identified by our model may influence transcription factor binding or nucleosomes indirectly, and could even affect mRNA translation. Transcription factors are critical for gene regulation. Their empirically identified binding sites are 6 to 8 bp, theoretically putting an upper bound on the level of regulatory flexibility that can be attained by mutating positions at these sites30,38. Cooperation between transcription factors or competition among them15–17, and with nucleosomes23, provides an additional mechanism for fine-tuned gene expression. RP promoters with high activity have not only more nucleosome disfavoring sequences but also characteristic spatial organization of the binding sites for Rap1, Sfp1 and Fhl130. The low performance of our model on synthetic promoters containing targeted mutations in transcription factor binding sites and nucleosome disfavoring sequences reinforces the importance of these factors. Consistent with this, the combination of our model and the mechanistically driven model involving transcription factors and nucleosome binding30 was more predictive of promoter activity34. Our findings have implications for understanding the fine-tuned regulation of RP genes and engineering desirable activity in synthetic promoters.\n\n\nData availability\n\nF1000Research: Dataset 1. Raw data for ‘Prediction of fine-tuned promoter activity from DNA sequence’, Siwo et al. 2016, 10.5256/f1000research.7485.d11351657", "appendix": "Author contributions\n\n\n\nGHS, RSP, AT, AKR conceived the methods and performed the analysis. All authors wrote the manuscript.\n\n\nCompeting interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThis work would not have been possible without the pre-publication provision of data to the DREAM challenge by Dr. Eran Segal and his group at the Weizmann Institute of Science, Israel, and the curation of the challenge by the DREAM committee: Drs. Gustavo Stolovitzky, Pablo Meyer and Rachel Norel at IBM Research, USA. 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Data Source" }	[ { "id": "12530", "date": "02 Mar 2016", "name": "Paul Pavlidis", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSiwo et al. give a detailed report of their entry to the DREAM promoter activity prediction assessment, conducted in 2011. The paper describing the results of the assessment appeared in 2013 (Meyer et al.), and the entry from Siwo et al. (“FiRST”) was the top-performer overall. Meyer et al. gives few details about the specific methods, mentioning only that the FiRST entry used an SVM and did not use TF binding site motif information. Here it is clarified that FiRST is a simple method that uses only part of the sequence and the most prominent features were about nucleotide content. Because it is perhaps a little eye-opening (even embarrassing, depending on one’s point of view) that the best method in the assessment is so simple, this paper is an important footnote to Meyer et al. but it could be fleshed out further to get at what is going on. My suggestions for revisions are to give more detail about the properties of the sequences used and the relationship to performance. FiRST predicts from only the 100 bases of sequence upstream of the translation start (which was considered as part of the promoter by DREAM; I note this is not “upstream of the gene” as described by Siwo et al. in the methods section), and that their predictions were dominated by the effect of a simple measure of G content. Siwo et al. report that they did worse at predicting the synthetically mutated promoters (this was apparently not true overall across methods as reported by Meyer et al.). In Meyer et al., adding tf binding information to FiRST improved performance. The authors mention this, but the most important reason that FiRST does poorly at predicting the synthetic mutations seems to be that most of the mutations (seems to be 29 out of 33, based on Table 1 of Meyer et al.) are not in the 100 bp window used. That is, because in most cases these synthetic sequences were (as I understand it) identical in features to other examples while having different activities, for the purposes of FiRST, they could only introduce prediction errors. In light of this fact the rest of the speculation about why performance varied in this way seems extraneous. It would also be useful to see more detailed information on the sequences used (e.g., the G content or other features), and the prediction error in each case. How well does one predict using G content alone? This might all be reconstructed from the data supplement helpfully provided, but the authors should consider providing the analysis. It also seems reasonable to ask for more details about the performance of other sequence windows. The main other missing piece from this paper is any discussion or evidence that the method works beyond the narrow confines of the DREAM setup. Even for the RP genes, does it make a useful prediction, that increasing the G content of RP promoters in that 100 bp window will decrease promoter activity? I am fine with leaving this as “future work” but it would be worth mentioning. Figure 2B is apparently the same as part of Figure 1E from Meyer et al., except FiRST is not marked (actually there is a small difference in the values plotted; the combined score for FiRST looks closer to 2 than the 1.87 reported and plotted in Meyer et al.). The authors should clearly cite Meyer et al. in the figure caption as the source of the data for this figure, or simply point the readers to Meyer et al., or else explain where the data came from if not from Meyer et al.", "responses": [] }, { "id": "12380", "date": "07 Apr 2016", "name": "Jianhua Ruan", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article describes the winning method of the DREAM6 promoter activity prediction challenge. While a meta analysis of the competing methods participated in the challenge has already been published (Meyer et al., 2013), this article provides more details of the winning method and some additional analysis of the predictive model, which may lead to better understanding of the predictability of gene transcription. While its contribution is undoubtable, this article should be revised to address several issues:Major issues:The 23 features utilized by the SVM model (as well as their coefficients in the model) is not provided explicitly in the main text nor in the supplement file. Table 1 in the main text shows that 6 trinucleotides and 12 tetranucleotides are important features, but it is nowhere to be found which tri- and tetra-nucleotides they are. For lengths of T or TA-tracts, the supplement file shows several different values, including mean, median and stdev. It is unclear which one is actually used by the SVM model. Similarly, supplement file shows 79 values for deformability and it is unknown which one is used. In the case of ranking the features by their SVM coefficients, the authors need to clarify if the feature values were normalized prior to model building, as these features are on very different scales and if not normalized the ranking of the coefficients are not very meaningful. The main conclusion in the subsection \"Error profile of SVM promoter activity model\" do not seem to make sense. First, promoters of low activity had larger prediction error. Then the authors stated that natural promoters had lower activity. This seems to contradict with their observation that the prediction error was significantly less for natural promoters than for mutated promoters.Minor issues:Authors only mentioned that feature selection was done in WEKA with wrapper. More details need to be given. For example, what was the selection strategy used by the wrapper, e.g., exhaustive search, greedy forward search, backward search, or other types of heuristics? What is the purpose of first training 1000 SVM classifiers using 66% of data as training and 34% as testing, and then another 500 SVM classifiers using 80% as training and 20% as testing?", "responses": [] }, { "id": "14225", "date": "20 Jun 2016", "name": "Jan Grau", "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 FIrST, an approach for predicting promoter activity from sequence, which won one of the DREAM6 challenges. FIrST is using only simple sequence features in a limited range (100 bp) upstream of the translation start site for making its predictions, which distinguishes it from several other approaches in this field. Prediction results are convincing and the method appears to be sound. However, currently the method is not described detailed enough. In addition, I have a few further major and minor concerns regarding the current version of the manuscript:\n\nMajor comments:\nIn the list of features described in section \"Feature extraction\", some seem redundant to me. For instance, the trinucleotide parameters for bendability are just computed from the k-mers for k=3. Also nucleosome binding prediction was based on trinucleotide preference. Please explain why it may be useful to also include those 3-mer-derived features in addition to the 3-mers themselves.\n\nThe description of methods in section \"Machine learning model exploration\" is too coarse. Please provide more detail on the SVMs, linear regression, and regression trees employed. It also remains unclear if the scales of features are normalized somehow, before their values are provided to the SVM.\n\nNo details are given on the selected 3-mers and 4-mers (Table 1). Please provide a list of the specific k-mers selected by FIrST. It may also be reasonable to discuss potential biological reasons for their importance (as partly covered for TATA-boxes on page 6).\n\nConsidering Fig. 3, I wondered if the difference in deformability may be related to transcription initiation. Or, stated differently, might we observe an ever clearer signal if all sequences (and their deformability profiles) would be aligned by the transcription start site (TSS) instead of the TrSS? One idea in the same direction, which could contribute to the novelty of the manuscript, would be to evaluate similar profiles (of sequences aligned to TSS or TrSS) for all features found to be informative by FIrST. For instance, one could expect to see something like general fluctuations of G/C content, or the TATA-box in 4-mer profiles as a spike approx. 35 bp before the TSS. From my perspective, this might improve the novelty of the manuscripts and the interpretation of features.\n\nMinor comments:\nThe data from the DREAM6 challenge only consider a special subset of genes (ribosomal genes) and only in yeast. It is unclear if the features derived by the authors' method would also be informative for higher eukaryotes. I understand that this question cannot be finally answered from the DREAM6 data, but the authors might comment on this issue.\n\nFigure 1B remains a bit unclear. In the caption and the main text, the authors explain that they use non-overlapping 100bp sub-sequences. However, from the figure it rather seems that they consider upstream sequences of 300 bp, 200 bp and 100 bp (and the full promoter sequence) relative to the translation start site. Please clarify.\n\nIn section \"DREAM6 challenge data\" of \"Methods\", the authors refer to \"the sequence 1200 bp upstream of a gene\", where \"upstream of the translation start site\" (as in the remainder of the text) would be more specific.\n\nIn section \"Feature extraction\", the authors explain that \"each promoter sequence was divided into 100 bp non-overlapping windows\", while in the previous section they explain that the full 1200 bp sequences do not extend over the nearest gene. From my understanding, this may result in some of the sequences being shorter than 1200 bp, and their length might not be dividable by 100. Please explain how such cases are handled.\n\nAt the end of section \"Validation of model by DREAM6 consortium\", the authors explain that \"the overall score was defined as the product of the four P-values\", whereas later they explain that -log10 of the geometric mean of the p-values was used as the overall measure. Although bot definition are equivalent with respect to the resulting ranking, I would suggest to provide one consistent definition of the overall score.\n\nFrom the manuscript it did not become fully clear if the TA-tracts (also termed poly(dA-dT) tracts in some parts of the manuscript) are tracts of poly \"A or T\" or tracts of poly \"AT\"-dinucleotides.\n\nIn section \"Error profile of SVM promoter activity model\", the authors explain that natural promoters had (slightly) lower activity than synthetic promoters and that the prediction error of the SVM is lower for natural promoters. However, I did not get the idea, why this should explain that low activity genes had larger prediction errors.\n\nIn section \"Error profile of SVM promoter activity model\", the authors explain that one reason why FIrST did not perform well for synthetic promoters is that most mutations had been introduced outside the 100 bp range considered by FIrST. However, this reasoning partly contradicts the claim of the authors that most of the transcriptional activity may be explained from the sequence in that 100 bp window. If this would truly be the case, mutations outside this range should have only minor effects.\n\nIn the Discussion, the authors mention that TF binding motifs are 6 to 8 bp in length. While this may be true for several yeast TFs, it is not correct for eukaryotes in general and motifs may be wider than 10 bp.", "responses": [] } ]	1	https://f1000research.com/articles/5-158
https://f1000research.com/articles/5-156/v1	10 Feb 16	{ "type": "Review", "title": "Continuous Attractor Neural Networks: Candidate of a Canonical Model for Neural Information Representation", "authors": [ "Si Wu", "K Y Michael Wong", "C C Alan Fung", "Yuanyuan Mi", "Wenhao Zhang", "K Y Michael Wong", "C C Alan Fung", "Yuanyuan Mi", "Wenhao Zhang" ], "abstract": "Owing to its many computationally desirable properties, the model of continuous attractor neural networks (CANNs) has been successfully applied to describe the encoding of simple continuous features in neural systems, such as orientation, moving direction, head direction, and spatial location of objects. Recent experimental and computational studies revealed that complex features of external inputs may also be encoded by low-dimensional CANNs embedded in the high-dimensional space of neural population activity. The new experimental data also confirmed the existence of the M-shaped correlation between neuronal responses, which is a correlation structure associated with the unique dynamics of CANNs. This body of evidence, which is reviewed in this report, suggests that CANNs may serve as a canonical model for neural information representation.", "keywords": [ "Continuous Attractor Neural Network", "Neural network", "multi-sensory information integration", "anticipative tracking", "canonical model" ], "content": "Introduction\n\nThe brain performs computation via dynamics of neural circuits formed by a large number of neurons. The dynamics of a neural circuit, on the other hand, are determined by the connection pattern between neurons. Thus, unveiling the structures of neural networks and their associated dynamical properties is at the core of elucidating brain functions. A question of common interest in both experimental and computational neuroscience is whether there exist canonical circuit models for neural information processing.\n\nOver the past few decades, a type of recurrent network, known as the continuous attractor neural network (CANN) or dynamic neural field, has received broad attention from computational neuroscientists1–3. This model has been successfully applied to describe the encoding of continuous stimuli in neural systems, such as orientation4, moving direction5, head direction6, and spatial location of objects7. The model has many computationally appealing properties, such as efficient population decoding8, smooth tracking of moving objects9, and implementing parametrical working memory10,11. The computational advantages of CANNs and their successes in modeling brain functions have suggested that CANNs serve as a canonical model for neural information representation. While there has been some evidence for CANN characteristics in the brain (e.g. the movement map in the superior colliculus)12, we review here recent important experimental findings and some new results for applying CANNs to modeling brain functions.\n\n\nThe model of CANNs\n\nThe CANN is a network model for neural information representation in which stimulus information is encoded in firing patterns of neurons, corresponding to stationary states (attractors) of the network. Compared with other attractor models, such as the Hopfield network13, the most prominent character of a CANN is its translation-invariant connections between neurons; that is, the connection strength between two neurons depends only on the difference between their preferred stimuli, rather than on the preferred stimulus values. This translation-invariant connection structure enables a CANN to hold a continuous family of attractors (stationary states), rather than isolated ones, with each of the attractor states encoding a stimulus value (this is where the name “continuous attractor” comes from). These states are often called bumps because of the localization of their activities in feature space. They form a submanifold of neutrally stable states in the state space of the network dynamics (see the illustration in Figure 1). This neutral stability endows a CANN with the capacity of updating its states (internal representations of stimuli) smoothly under the drive of an external input. Several mathematical formulations for CANNs have been proposed in the literature. Here, for convenience of description, we present the one whose dynamical behaviors are analytically solvable14,15, although the dynamical behaviors of many other models are similar.\n\n(A) An illustration of a one-dimensional CANN, which encodes a continuous variable (e.g. orientation or direction) x in the region of (-π,π] with the periodic condition. Neurons are aligned in the network according to their preferred stimuli. The neuronal connection pattern J(x,x’) is translation-invariant in the space. The network can hold a continuous family of bump-shaped stationary states. (B) The stationary states of the CANN form a subspace in which the network states are neutrally stable. The subspace is illustrated as a canyon in the state space of the network. The movement of the network state along the canyon corresponds to the position shift of a bump.\n\nConsider a one-dimensional continuous stimulus x, such as head-direction or orientation, encoded by an ensemble of neurons, and the value of x is in the range of (-π,π] with a periodic boundary. In the space of stimulus x, neurons are aligned in the network according to their preferred stimulus values. Denote U(x,t) as the synaptic input at time t of the neurons whose preferred stimulus is x, and r(x,t) the neuronal firing rate. The dynamics of U(x,t) are determined by the recurrent input from other neurons, its own relaxation, and an external input Iext(x,t), which is written as,\n\n\n\nwhere τ is the synaptic time constant and ρ the neuron density. J(x,x′) is the interaction strength from neurons at x′ to neurons at x, and is chosen to be J(x,x′)=J0/(2πa)exp⁡[−(x−x′)2/2a2], where the parameter a controls the neuronal interaction range. Note that J(x,x′) is a function of (x – x′); that is, the neuronal interaction is translation-invariant in the space of neuronal preferred stimuli. The neuronal firing rate r(x, t) is determined by the synaptic input according to\n\n\n\nwhere [U]+ ≡ max(U, 0). The neuronal firing rate first increases with the input and then saturates gradually because of divisive normalization by the total network activity. In the absence of external input and for 0<k<kc≡ρJ02/(82πa), the network holds a continuous family of stationary states, which are written as U¯(x\|z)=U0exp⁡[−(x−z)2/(4a2)] and r¯(x\|z)=r0exp⁡[−(x−z)2/(2a2)]. These stationary states are translationally invariant and have a Gaussian-bump shape with a free parameter z indicating their positions.\n\nThe dynamical behaviors of a CANN can be readily analyzed by a projection method14 by using the property that the dynamics of a CANN are dominated by a few motion modes, which correspond to distortions of the bump shape in terms of height, position, width, skewness, and so on in the bump shape (Figure 2). We can project the dynamics of a CANN onto these dominating modes and simplify the network dynamics significantly. Typically, by including one or two leading motion modes, the simplified dynamics are adequate to capture the main features of a CANN.\n\nThe dynamics of a continuous attractor neural network are dominated by a few motion modes, corresponding to distortions of the bump shape in height, position, width, skewness, and so on. We can project the network dynamics on these dominating modes to simplify it significantly.\n\n\nComputational advantages of CANNs\n\nA large volume of theoretical studies revealed many computationally appealing properties of CANNs (see, for example, 2,3,8,15). Here, we present two that were recently proposed in the literature.\n\n\nCANNs for anticipative tracking\n\nTime delays are pervasive and significant in neural information processing; for example, visual signal transmitting from the retina to the primary visual cortex (V1) takes about 40 to 80 ms16. If these delays are not compensated properly, our perception of a fast-moving object will lag behind its true position in the visual world significantly, impairing our vision and motor control. A CANN is able to track a moving object smoothly. However, its reaction is always lagging behind the object location because of the time needed for neuronal responses and neuronal interactions. In recent studies, Fung et al.17 and Mi et al.18 found that, by incorporating slow negative feedback modulation in the network dynamics, a CANN is able to achieve anticipative tracking, compensating for delays in neural systems (Figure 3A). The negative feedback modulation can be realized by a number of mechanisms, including spike-frequency adaptation in neuronal firing18, short-term depression of neuronal synapses19, or negative feedback from a connected network20. Since the negative feedback reduces the firing rate at the peak of the bump but is weaker at the shoulder, the tendency of the bump to move to its vicinity is enhanced. This increases its mobility, and the bump can move spontaneously when the negative feedback is sufficiently strong. Remarkably, the parameter region of spontaneous motion effectively coincides with the region of anticipative tracking (Figures 3C and 3D). Different models relying on the asymmetric connections between neurons in a CANN were proposed to generate anticipative neural responses (see, for example, 6,21). Here, the mechanism based on the negative feedback modulation has the advantage of realizing a constant anticipative time irrespectively of the object speed (Figure 3B), agreeing with the experimental finding on the anticipative behavior of head-direction neurons in rodents22.\n\n(A) In the presence of spike-frequency adaptation (SFA), the network bump (red solid curve) leads the external input (blue dotted curve) moving with velocity Vext. Without SFA, the bump (green dashed curve) lags behind the external input. Inset: the positions of the bumps as a function of time when the external input starts to move at a constant velocity after t = 0. (B) The anticipative time tant is approximately constant in a broad range of Vext. Symbols represent anticipative time from Goodridge and Touretzky22 rescaled for comparison. (C) Static and spontaneously moving phases in the space of inhibition strength k˜≡k/kc and SFA strength γ. The black curve indicates the phase boundary separating the static and moving phases. In the moving phase, the color code encodes the speed Vint of the spontaneously moving bump. (D) Regions of delayed and anticipative tracking in the same space when there is a weak and slowly moving external input. The black curve indicates the boundary separating the delayed and anticipative tracking regions. The color code encodes the anticipative time (negative values indicate delayed time). Note the correspondence between the anticipative time and Vint in (C).\n\n\nCANNs for multi-sensory information integration\n\nThe brain exploits multiple sensory modalities to gather, from different channels, as much information as possible about the surrounding environment. Psychophysical studies reveal that the brain can integrate these different sensory cues optimally to improve its perception23. However, exactly how the brain achieves this remains largely unknown. In a recent study, Zhang and Wu24 proposed that the brain may employ a decentralized architecture with coupled CANNs to carry out this task. In their model, multiple CANNs, each corresponding to one sensory module, are reciprocally connected with each other, and the connection strengths control the extent of integration (Figure 4A). Mediated by reciprocal interactions, information from different cues is exchanged between sensory modules, such that global information integration is achieved at each local processor without the need for a centralized integration unit. By applying this model to the visual and vestibular cue integration for inferring heading direction, Zhang and Wu24 showed that the decentralized architecture with coupled CANNs can explain a large volume of data about the integration behaviors observed in the multi-sensory experiments (Figures 4B–4D).\n\n(A) Multiple reciprocally coupled CANNs form a decentralized information integration system. (B) An example of two-coupled CANNs for heading-direction inference with combined visual and vestibular cues. The mean (C) and the variance (D) of the network estimations agree with the Bayesian predictions. Adapted from 24.\n\n\nNeural signature of CANNs\n\nThe key structure of a CANN is the translation invariance of the connections between neurons. Limited by experimental techniques, we are still unable to confirm the existence of such a connection pattern of synapses in real neural systems. Nevertheless, we can validate the existence of a CANN by measuring its unique dynamical features. One such feature is the anti-symmetric, or the M-shaped, correlation between neuronal responses15,25. The underlying cause is intuitively understandable. The dynamics of a CANN are dominated by the position shift of a bump under the drive of noisy inputs. Thus, in response to a stimulus corrupted with noise, neurons whose preferred stimuli are on the same side of the true stimulus (i.e. they are both larger or both smaller than the stimulus value, as illustrated in Figure 5A) will increase or decrease their responses concurrently with the fluctuations of the bump position, leading to a positive correlation, whereas for neurons whose preferred stimuli are on different sides of the stimulus, their response fluctuations are negatively correlated (Figure 5B). Alternatively, we can measure the correlations of firing rates between a pair of neurons with a typical separation of the bump width in a CANN by varying the stimulus value systematically. In such a case, we obtain the M-shaped correlation over the stimulus value space: when the stimulus value is in between the preferred stimuli of two neurons, the neuronal responses are negatively correlated; otherwise, they are positively correlated (Figure 5C).\n\nNotably, the M-shaped correlation between a neuron pair in the cortex was confirmed in recent experiments. In the study, Wimmer et al.26 used multiple electrodes to record the activities of neurons in the prefrontal cortex (PFC) of monkeys when they were performing a working memory task. The monkeys were firstly presented with a stimulus appearing randomly in one of eight possible directions, and then the monkeys needed to memorize the stimulus location during a delay period when the stimulus was off. Wimmer et al. found that the dynamics of a CANN can well explain the behaviors of the monkey and that the correlation between PFC neurons in the delay period is M-shaped. In another study, Ponce-Alvarez et al.25 measured the correlation between a neuron pair in the middle temporal area when monkeys were presented with moving grating or plaid, and also found the M-shaped structure.\n\n(A) The bump position-shift is the dominating motion mode of a CANN induced by input noises. Consider the true stimulus fixed at zero. Neurons at the same side of the stimulus are positively correlated (e.g. green ones), whereas neurons on different sides of the stimulus are negatively correlated (e.g. green versus blue ones). (B) When the true stimulus is fixed at a constant value (e.g. zero), the correlations between all neuron pairs in a CANN display an anti-symmetric structure. (C) When the stimulus value varies, the correlation between a fixed neuron pair with a typical separation of the bump width displays an M-shaped structure.\n\n\nBeyond simple features\n\nIn addition to the aforementioned simple, straightforward features of objects, such as orientation, direction, and spatial location, experimental data suggest that the brain may use CANNs to process less directly perceivable features. For example, in an experimental study, Logothetis et al.27 found that, after training, neurons in the inferior temporal cortex of the monkey’s brain displayed strong selectivity to the view angle of an object and that the neuronal tuning functions were of the bell shape and were aligned to cover the space of view angles, similar to the structure of a CANN. Furthermore, the brain may use CANNs to process “complicated” features. In recent work, Mante et al.28 studied the dynamical properties of neuronal responses in the PFC when monkeys executed a context-dependent choice task, in which the monkeys made a left or right saccade depending on a flexible context cue, which was either direction of motion or color. Interestingly, the authors found that although individual neurons’ responses exhibited intractable complexity, the neural responses at the population level follow a low-dimensional trajectory embedded in the high-dimensional space. Mante et al. further trained a network model to interpret the experimental finding and found that the CANN structure automatically emerged in the trained network: the network held a set of stationary states forming the canyon of a one-dimensional CANN, integration of cue evidence was implemented in the model as movement along the canyon (approximately), and different strengths of sensory inputs led to different stationary states in the canyon. The CANN model well reproduced the experimental data, indicating that PFC neurons may exploit the CANN structure to encode the subject value of evidence/confidence in decision making.\n\n\nFurther aspects\n\nIn conclusion, the accumulated facts, including the computationally appealing properties, many successful examples in modeling brain functions, and the new supporting experimental data, suggest that CANNs may serve as a canonical model for information representation in neural systems. Nevertheless, there is still a lot of work to do to validate this hypothesis. In experiments, the vast development of imaging techniques will eventually give us direct evidence of whether neuronal synapses are translation-invariant in a feature space and, if they are, where in the brain and to what extent. In theory, as implied by the above-reviewed work, we should explore deeply the functional roles of CANNs, including how CANNs contribute to the information exchanges constantly happening between cortical areas, how the CANN structure emerges automatically via either supervised or unsupervised learning in a given computational task, and how a CANN, which has the capacity of encoding complex, non-trivial features of external inputs (as suggested by the work of Mante et al.), contributes to the categorization of objects or formulation of concepts. Overall, these studies will not only enhance our understanding of the principles of neural information processing but also reveal more computational advantages of CANNs which are useful for developing brain-inspired computation algorithms.", "appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThis work is supported by grants from National Foundation of Natural Science of China (31261160495, SW; 11305112, YYM), and Research Grants Council of Hong Kong (605813, 604512, KYW).\n\n\nReferences\n\nAmari S: Dynamics of pattern formation in lateral-inhibition type neural fields. Biol Cybern. 1977; 27(2): 77–87. PubMed Abstract \| Publisher Full Text\n\nErmentrout B: Neural networks as spatio-temporal pattern-forming systems. Rep Prog Phys. 1998; 61: 353–430. Publisher Full Text\n\nBressloff PC: Spatiotemporal Dynamics of Continuum Neural Fields. J Phys A. 2012; 45(3): 033001. 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PubMed Abstract \| Publisher Full Text\n\nMi Y, Fung CA, Wong KYM, et al.: Spike Frequency Adaptation Implements Anticipative Tracking in Continuous Attractor Neural Networks. Paper presented at: Advances in Neural Information Processing Systems. 2014. Reference Source\n\nFung CC, Wong KY, Wang H, et al.: Dynamical synapses enhance neural information processing: gracefulness, accuracy, and mobility. Neural Comput. 2012; 24(5): 1147–1185. PubMed Abstract \| Publisher Full Text\n\nZhang W, Wu S: Neural information processing with feedback modulations. Neural Comput. 2012; 24(7): 1695–1721. PubMed Abstract \| Publisher Full Text\n\nS Fard F, Hollensen P, Heinke D, et al.: Modeling human target reaching with an adaptive observer implemented with dynamic neural fields. Neural Netw. 2015; 72: 13–30. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nGoodridge JP, Touretzky DS: Modeling attractor deformation in the rodent head-direction system. J Neurophysiol. 2000; 83(6): 3402–3410. PubMed Abstract \| Faculty Opinions Recommendation\n\nErnst MO, Banks MS: Humans integrate visual and haptic information in a statistically optimal fashion. Nature. 2002; 415(6870): 429–433. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nZhang W, Wu S: Reciprocally Coupled Local Estimators Implement Bayesian Information Integration Distributively. Paper presented at: Advances in Neural Information Processing Systems. 2013. Reference Source\n\nPonce-Alvarez A, Thiele A, Albright TD, et al.: Stimulus-dependent variability and noise correlations in cortical MT neurons. Proc Natl Acad Sci U S A. 2013; 110(32): 13162–13167. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nWimmer K, Nykamp DQ, Constantinidis C, et al.: Bump attractor dynamics in prefrontal cortex explains behavioral precision in spatial working memory. Nat Neurosci. 2014; 17(3): 431–439. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nLogothetis NK, Pauls J, Bülthoff HH, et al.: View-dependent object recognition by monkeys. Curr Biol. 1994; 4(5): 401–414. PubMed Abstract \| Publisher Full Text\n\nMante V, Sussillo D, Shenoy KV, et al.: Context-dependent computation by recurrent dynamics in prefrontal cortex. Nature. 2013; 503(7474): 78–84. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation" }	[ { "id": "12341", "date": "10 Feb 2016", "name": "Yi Zhang", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12340", "date": "10 Feb 2016", "name": "Thomas Trappenberg", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-156
https://f1000research.com/articles/4-457/v1	03 Aug 15	{ "type": "Research Article", "title": "EEG correlates of social interaction at distance", "authors": [ "William Giroldini", "Luciano Pederzoli", "Marco Bilucaglia", "Patrizio Caini", "Alessandro Ferrini", "Simone Melloni", "Elena Prati", "Patrizio E. Tressoldi", "William Giroldini", "Luciano Pederzoli", "Marco Bilucaglia", "Patrizio Caini", "Alessandro Ferrini", "Simone Melloni", "Elena Prati" ], "abstract": "This study investigated EEG correlates of social interaction at distance between twenty-five pairs of participants who were not connected by any traditional channels of communication.Each session involved the application of 128 stimulations separated by intervals of random duration ranging from 4 to 6 seconds. One of the pair received a one-second stimulation from a light signal produced by an arrangement of red LEDs, and a simultaneous 500 Hz sinusoidal audio signal of the same length. The other member of the pair sat in an isolated sound-proof room, such that any sensory interaction between the pair was impossible.An analysis of the Event-Related Potentials associated with sensory stimulation using traditional averaging methods showed a distinct peak at approximately 300 ms, but only in the EEG activity of subjects who were directly stimulated. However, when a new algorithm was applied to the EEG activity based on the correlation between signals from all active electrodes, a weak but robust response was also detected in the EEG activity of the passive member of the pair, particularly within 9 – 10 Hz in the Alpha range. Using the Bootstrap method and the Monte Carlo emulation, this signal was found to be statistically significant.", "keywords": [ "mental entanglement", "EEG", "ERP", "bootstrap", "Monte Carlo." ], "content": "Introduction\n\nThe study of EEG correlates of social interaction is a hot topic in the world of social neuroscience, described as follows by Cacioppo & Bertson (2002): “Social neuroscience addresses fundamental questions about the mind and its dynamic interactions with the biological systems of the brain and the social world in which it resides”.\n\nThe study of EEG correlates of social interaction ranges from simple face-to-face motor interactions (e.g. Hari et al., 2013), to empathy (Singer & Lamm, 2009), to interpersonal motor co-ordination (Oullier et al., 2008; Sebanz & Knoblich, 2009). A recent review of the current status of the field, particularly in reference to social cognition, is given by Chatel-Goldman et al. (2013). This review highlights the importance of not underestimating possible non-local mechanisms that can emerge from person-to-person interactions. These mechanisms are defined as “dependent operations between two or more brains that operate at least in part on shared information content” and are also described as interactive alignment, resonance, phase synchronization, and non-local correlations.\n\nIs it conceivable that these mechanisms can be detected even when two persons are mentally interacting without the possibility of sensory information exchange?\n\nThis possibility is rarely studied, not so much because of technical or methodological difficulties, but because the prevailing view is that the human mind can only receive information through the five senses and anything else is impossible. Nonetheless, if we assume that the human mind is also capable of receiving and processing information transmitted from other than the five senses, it becomes possible to investigate the characteristics of mental activity related to the interaction between two sensorily isolated individuals.\n\nThis model of mind function, also defined as “non-local” because it is not limited to the spatial and temporal confines of the five senses, is predicted by various theoretical models. For example, according to Dual-Aspect Monism (Atmanspacher, 2012), there is neither a material reality nor a mental reality – rather, they are two different aspects of one reality. These mental characteristics are also consistent with Generalized Quantum Theory (GQT) proposed by Walach & von Stillfried (2011), and Filk & Römer (2011). This theory postulates the existence of a distant mind-to-mind connection (mental entanglement), similar to the entanglement phenomena of quantum physics.\n\nAccording to these authors: “The genuinely quantum theoretical phenomenon of entanglement can and in general will show up also in GQT if the following conditions are fulfilled:\n\n1) A system is given, inside which subsystems can be identified. Entanglement phenomena will be best visible if the subsystems are sufficiently separated such that local observables pertaining to different subsystems are compatible.\n\n2) There is a global observable of the total system, which is complementary to local observables of the subsystems.\n\n3) The total system is in an entangled state. For instance, eigenstates of the global observable are typically entangled states.\n\nA distant social interaction between two persons who know each other satisfies these requirements provided that:\n\na) the two persons represent two subsystems of a single larger one created by their relationship, and\n\nb) this relationship constitutes an entangled state, and furthermore that\n\nc) the measurable psychological and physiological variables represent the system’s comprehensive characteristic even though measured individually.\n\nThe study presented here is a further addition to this field of research. In comparison to other research, our specific objectives are:\n\n1) To determine the difference in power or other statistical characteristics of the EEG signal between the person receiving the physical stimulus and his/her mentally connected partner;\n\n2) Determine the latency period, if any, between the EEG signals and the stimulus of both partners;\n\n3) Determine the frequency ranges of EEG activity that best represent the connection between the subject pair.\n\n\nMethods\n\nSix Italian Caucasian healthy adults were chosen for the experiment, comprised of five men and one woman, with an average age of 35.5 years (standard deviation = 8.3).\n\nThey were selected among the members of the EvanLab, the private laboratory involved in this study. The criteria for their voluntary inclusion were their mutual friendship (> 10 years), and their experience in being able to maintain prolonged focused concentration – a product of their familiarity with meditation and other practices requiring control of mental activities.\n\nThe use of experimental subjects is in accordance with ethical guidelines as outlined in the Declaration of Helsinki, and the study has been approved by the Ethical Committee of the University of Padova’s Department of General Psychology. Before taking part in the experiment, each subject gave his/her informed consent in writing after having read a description of said experiment.\n\nA software program, available at http://dx.doi.org/10.6084/m9.figshare.1466876, especially written by one of our co-authors (GW) administered the sequence of stimuli and synchronized the EEG recordings from each member of the pairs. EEG activity was measured using two Emotiv® EEG Neuroheadsets, equipped with 14 EEG channels, connected via WiFi to a Windows PC.\n\nThe technical details are: 14 electroencephalography channels based on international location from 10 to 20 (AF3, F7, F3, FC5, T7, P7, O1, O2, P8, T8, FC6, F4, F8, AF4, plus two reference electrodes). The mastoid electrodes (M1, M2) served as reference points against which the voltage generated from all other electrodes was compared. The sample frequency of the Emotiv® headsets is 128 Hz, with a bandwidth from 0.2 to 45 Hz, with a built-in fifth order low-pass digital filter as well as two notch filters at 50 and 60 Hz respectively as protection against noise produced by the local electricity network. The Emotiv® EEG has a proprietary wireless network connection at a frequency of 2.4 GHz.\n\nThe auditory stimulus was composed of a 500 Hz sinusoid applied through 32 Ohm Parrot ZIK® earphones at a volume of about 80 dB. The visual stimulation was from high intensity red LEDs in a 4×4 arrangement placed approximately one meter from the subject being stimulated. The subject kept his/her eyes closed because the light could easily be detected through the eyelids.\n\nThe members of each pair were placed in two separate rooms approximately five meters from each other. Each room was sound- and light-proof, so as to block out any and all external sensory information.\n\nBetween these two rooms was a control room with two computers connected to the Emotiv® headsets and from which the research assistant controlled the sensory stimulation program and each partner’s EEG recording (see Figure S1 in Supplementary material). The software program in use ensured that the signals coming from the two EEG headsets were recorded simultaneously (to within 8 ms).\n\nThe partner designated as “Sender” was given the following instructions: “When you are ready, relax and be prepared to receive a visual and auditory stimulus which you will send to your partner. To assist your mental connection with him/her, concentrate on his/her photo before starting the experiment. Your only task is to mentally transmit what you will perceive, while limiting your body movements to prevent interference with your EEG activity. You will perceive 128 stimulations of 1 second each, separated by pauses of random length lasting 4 to 6 seconds in order to avoid predictable rhythms. The experiment will last about 10 minutes.”\n\nThe partner designated “Receiver” was given the following instructions: “When you are ready, relax and be prepared to receive the stimuli sent from your partner. To assist your mental connection with him/her, you will see a facial photo of him/her before starting the experiment. Your task is to mentally connect with him/her and try to perceive the stimulus he/she is receiving, while keeping your body still to prevent interference with your EEG activity. The experiment will last about 10 minutes.”\n\nOnce the quality of the EEG signals was confirmed, and with the consent of the subjects, the research assistant began running the experiment’s program. To prevent either subject from predicting when the first stimulus would be given, it was preceded by a period of silence of random duration from 2 to 3 minutes.\n\nAt the end of the experiment, after a period of rest, in m ost cases (if subjects agreed and time allowed) the role of each subject was reversed.\n\nAll together data from 25 pairs of subjects was collected over three days. The raw data are available at http://dx.doi.org/10.6084/m9.figshare.1466876 which include details of pairings.\n\nTo avoid any experimenter’s effect, the research assistant who managed the software for the data acquisition were blind to the exact start of the stimulation sequence, given the randomization of the duration of the first pre-stimulation period as described above.\n\nThe reduction of the risk of any conventional communication between the pair of participants, was guaranteed by the sensory isolation of the two rooms were they were placed as already described. The only remaining possibility was to speak aloud each other, but this event could clearly be noticed by the research assistant.\n\n\nResults\n\nCollection of the evoked potential was initially conducted by filtering the signals in the 1 – 12 Hz band followed by normalization (see software code of Appendix 1 at http://dx.doi.org/10.1101/022046), hence employing the traditional averaging method of time- and phase-locked epochs. The typical result of an evoked potential obtained from Senders can be seen in the graph in Figure 1.\n\nThe graph is an average of 128 stimuli and 14 EEG channels. Usually two peaks are seen, a negative and a positive one, about 250 to 300 ms after the stimulus begins, and a minor peak at about 250 ms after the stimulus ceases.\n\nThe average Event-Related Potential (ERP) of all 25 files from Senders and Receivers was also calculated. To get the total sum of evoked potentials from all subjects while avoiding ERP different latency period problems, each subject’s individual evoked potential powers were added up. The resulting graph is shown in Figure 2.\n\nFigure 2 clearly shows an ERP in the Senders, but nothing of interest in Receivers.\n\nFollowing this negative result we began using an original method created by one of our co-authors (WG), and described in detail in Tressoldi et al. (unpublished; pre-print proof available at http://biorxiv.org/content/early/2015/07/06/022046), which was found more resistant to jitter and interferences compared to the traditional averaging method. Furthermore, as described below and in more detail in the original paper, this new processing method is far less prone to unwanted effects of EEG artifact because as the Pearson Correlation depends only on signal phase and not to amplitude.\n\nThis method is based on the Pearson correlation between segments of data of fixed length L, as shown in Figure 3.\n\nThe corresponding Pearson Correlation is calculated and recorded on the R(x) array.\n\nAs an example, the Emotiv® EEG Neuroheadset provides NC = 14 EEG channels and a sample frequency of 128/s; the stimulus is 1 second duration and an epoch’s length is 3 seconds, equal to 384 samples. In this case it becomes possible to calculate the R(x) array in a number of combinations of pairs equal to: Nt = NC*(NC - 1)/2 = 91. The result can be written using a new array, R(I, X), in which I = 1... 91 and X = 1... 384 are the calculated values. The stimulus is administered at the same time as sample no. 128 and ceases after one second, with sample no. 256. The next processing step involves the average of R(I, X) over all the given stimuli, the result designated as R’(I, X).\n\nTherefore, for each value of I = 1.... 91 a baseline is calculated, comprised of the average of R’(I, X) in the pre- and post- stimulus areas. Finally a new array is calculated, Sync(I, X), based on the result’s absolute value: Sync(I, X) = Abs(R’(I, X-Baseline)).\n\nThen the average of all the Nt combinations gives us the final array, Sync1(X), which represents the total variations of the EEG correlations during a 3 second epoch, for all stimuli and all EEG channels. It is also possible to calculate a similar array, Sync2(C, X), for each channel, C.\n\nTo be extra certain, the analysis of experimental data was nonetheless conducted on longer epochs – up to 4 seconds – comprised of 1.5 seconds pre-stimulus, 1 second stimulus, and 1.5 seconds post-stimulus. To calculate the probability that the observed differences in baselines are due to chance, the experimental data were compared to those obtained with a simulation conducted using a bootstrap procedure with the following characteristics:\n\na) Signals from the desired frequency range (in our case 9–10 Hz in Receivers and 1–16 Hz in Senders) are filtered using a digital filter that leaves signal phases intact, according to the Discrete Fourier Transform (DFT) and its related inverse processing. The filtered files are then saved. We point out that for Senders the standard frequency range (1–16 Hz) was used because the ERP is usually generated in this range.\n\nb) The same processing method (The GW6 method) is applied to these files, but choosing at random the point in which a stimulus is thought to be present. For each file, the same number of stimuli (128) are evaluated, as in the experimental tests. For each file at least 20 bootstrap calculations are made, eventually resulting in over 500 files. The average of these calculations constitutes the blue bootstrap curve in Figure 4 and Figure 5, which therefore represents the expected probability due to chance, to compare with the obtained experimental curves (red). This method appears valid in that it gives a virtually flat curve (blue), close to zero throughout.\n\nOn the vertical axis are correlation values x100. The blue curve denotes the average of 500 bootstrap files.\n\nThe red curve is the average correlation and the blue is the bootstrap curve (average of 500 files), which represents the expected probability due to chance.\n\nIn Figure 4, a distinct peak (red curve) is seen which represents a correlation ERP at about 300 ms from the start of the stimulus, followed by a weaker peak at the end of the stimulus. This graph is similar to the one obtained from standard averaging in Figure 2, only expanded.\n\nIn Figure 5, with respect to the Receivers, there is an area that exceeds the normal chance expectation represented by the bootstrap curve. This area is highlighted in yellow and can be calculated as the difference with respect to the bootstrap curve.\n\nA similar analysis was conducted by filtering signals within the 1 to 16 Hz band (as in the Senders) with statistically null results, and subsequently in the 8 to 16 Hz band, followed by the 8 to 12 Hz, but the best result was obtained in the 9 to 10 Hz range (see Table 1).\n\nThe column on the right shows the probabilities that the results are purely due to chance.\n\nAfter having established an increase in cerebral correlation in Receivers coinciding with the remote stimulus given to Senders, it is necessary to determine the importance of this difference with respect to the statistical chance. To this end, instead of resorting to conventional statistical methods, often inapplicable to complex situations such as this, we used an emulation procedure of the Monte Carlo type consisting of the following steps:\n\na) Take the bootstrap files within the desired frequency band – in our case from 9 to 10 Hz (around 500 files), or from other bands. All of these files are the result of a GW6-type processing and are now the input data set for the Monte Carlo emulation.\n\nb) 25 “fake” files are randomly chosen and their final average is calculated, in the same way as the average of the 25 “real” files.\n\nc) The difference in area with respect to the average of all the 500 bootstrap curves is calculated, as in Figure 5. This difference may be either a negative or positive number.\n\nd) Determine if this number is higher than that obtained from the real experimental files. If it is higher, a counter is incremented.\n\ne) Start again from a) and repeat the cycle as required (we repeated the cycle 2000 times).\n\nAt the end, determine how many times out of 1000 a group of 25 bootstrap files randomly exceeds the value of the area found experimentally. The results give the probability of obtaining a surplus of area by chance, as in Figure 6. This procedure does not use any specific a priori statistical model, and is based solely on applying numerous emulations exactly as with real data. As shown in Table 1, in the 9 to 10 Hz band the result is significant to a level of around 2–3/1000, equivalent to P ≤ 0.003, and is even significant in the 8 to 12Hz band, with P ≤ 0.04. The almost Gaussian distribution of the values shows that the method is valid and agrees with normal statistics.\n\nThe value of the area to be exceeded (see Table 1) is 0.3106 (red line). The distribution is approximately Gaussian and the specified value is exceeded by chance 3 out of 2000 times.\n\n\nDiscussion\n\nWhen traditional methods of averaging and calculations for ERP power are used, there is a distinct evoked potential in the EEGs of subjects who are Senders, but not in the EEGs of Receivers.\n\nConversely, when the GW6 method is used and the signals within the 9 – 10 Hz band are filtered, we obtain the results shown in Figure 5, which are statistically confirmed by the Monte Carlo Emulation. These outcomes lead us to believe that Receivers exhibit a weak response to the remote stimulus in the form of a small change in cerebral synchronization coinciding with the stimulus. This variation approximately equates to a 0.5% correlation, with a maximum of about 1.5–2.0% in the best subjects under examination. Even though the applied method does not display a result in the form of a wave similar to that seen in the Senders’ ERPs, this result does however open the door to future investigations aimed at identifying specific patterns of weak but significant responses in Receivers. This study is clearly explorative but if our results were confirmed, they would be of huge scientific importance because they provide neurophysiological evidence of a connection – or social interaction – at distance.\n\nRegarding future developments in this area, we will attempt to identify EEG signals in Receivers while applying a gradual reduction in the number of stimulations. Continual advances in techniques for processing EEG signals allow us to be optimistic in reaching this objective.\n\n\nData availability\n\nThe raw dataset and software codes for this article are available at: http://dx.doi.org/10.6084/m9.figshare.1466876\n\nThe unpublished proof describing the GW6 method is available at: http://biorxiv.org/content/early/2015/07/06/022046", "appendix": "Author contributions\n\n\n\nGW, LP and SM conceived the study and designed the experiments. All authors carried out the research. GW, LP and PT 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\nWe kindly acknowledge the support of the BIAL Foundation, which funded part of this study through grant no. 124/12.\n\nWe confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThanks to C. Evangelista-Pannozzo (Melbourne) for the English translation.\n\n\nSupplementary material\n\n\n\n\nReferences\n\nAtmanspacher H: Dual-aspect monism a la Pauli and Jung. J Conscious Stud. 2012; 19(9–10): 96–120. Reference Source\n\nCacioppo JT, Berntson G: Social Neuroscience. In Cacioppo JT (Ed). Found Soc Neurosci. 2002; MIT Press.\n\nChatel-Goldman J, Schwartz JL, Jutten C, et al.: Non-local mind from the perspective of social cognition. Front Hum Neurosci. 2013; 7: 107. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFilk T, Römer H: Generalized quantum theory: Overview and latest developments. Axiomathes. 2011; 21(2): 211–220. Publisher Full Text\n\nHari R, Himberg T, Nummenmaa L, et al.: Synchrony of brains and bodies during implicit interpersonal interaction. Trends Cogn Sci. 2013; 17(3): 105–106. PubMed Abstract \| Publisher Full Text\n\nOullier O, de Guzman GC, Jantzen KJ, et al.: Social coordination dynamics: Measuring human bonding. Soc Neurosci. 2008; 3(2): 178–192. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSebanz N, Knoblich G: Prediction in joint action: what, when, and where. Top Cogn Sci. 2009; 1(2): 353–367. PubMed Abstract \| Publisher Full Text\n\nSinger T, Lamm C: The social neuroscience of empathy. Ann N Y Acad Sci. 2009; 1156(1): 81–96. PubMed Abstract \| Publisher Full Text\n\nTressoldi P, Giroldini W, Pederzoli L, et al.: A new method to detect Event-Related Potentials based on Pearson's correlation. bioRxiv. Publisher Full Text\n\nWalach H, von Stillfried N: Generalised Quantum Theory - basic idea and general intuition: a background story and overview. Axiomathes. 2011; 21(2): 185– 209. Publisher Full Text" }	[ { "id": "9814", "date": "10 Sep 2015", "name": "Thilo Hinterberger", "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 reports a study in which spatially separated pairs of participants have been measured with EEG simultaneously. One of them was audiovisually stimulated at random times. The hypothesis was tested whether an evoked response related to the stimulation in one participant could be detected in the EEG of the non-stimulated participant. A nonparametrical analysis using bootstrapping analysis showed a positive effect in the alpha frequency range.The manuscript is written clearly and the experiment and analysis seem to be done in a scientific rigor way. However, several questions arose and some additions and changes should be done before full approval of this paper. These are described in the following:This type of experiment was already done and published by various researchers. Almost none of them is mentioned but I think this is important as the study is a kind of replication of previous studies. Especially, as there are experiments that showed an effect in the alpha band which supports your findings. I would suggest to report previous studies of this type in the introduction and shortly report their findings (E.g.by Wackermann, Radin, Hinterberger,…). In the introduction the Generalized Quantum Theory is presented as a rational for the hypothesis. I would be very careful with this approach because it is not clear whether this study design can be used to test the WQT and further whether the WQT actually applies to the phenomenon of distant mental interactions even if it is an elegant model. Spoken as a physicist, I do not really see the conditions for entanglement fulfilled because not every subsystem, that has information of another subsystem and shares the idea of interconnectedness behaves as being entangled. Therefore, it seems to be oversimplified to just state that \"this relationship constitutes an entangled state\".Another problem arises with the non-signal transfer paradigm which in this type of experiment would be violated if we would find replicable correlations of the same type. In such case one should find a different explanation for the effect.Therefore, I suggest instead of claiming the WQT in the introduction, the study should be presented as a replication of previous studies in a slightly different manner, testing an experiential phenomenon reported by many people. Also please state why you were using this type of stimuli. The WQT would probably fit better as an attempt for an explanation of the findings in the discussion or, if used in the introduction with much more care. Why were you focusing on the frequency range between 9-10 Hz? Was this a post-hoc selection? Could you tell the results of the other standard frequencies such as theta, beta,.. bands? “…it becomes possible to calculate the R(x) array in a number of combinations of pairs equal to: Nt = NC*(NC - 1)/2 = 91. The result can be written using a new array, R(I, X), in which I = 1... 91 and X = 1... 384 are the calculated values.“This description remains unclear to me. Could you describe the meaning of the variables and explain the process a bit more? What is the GW6 method? Is it possible to tell something about the localization of your findings as you recorded 14 channels? In the discussion please compare your findings to those of similar studies.", "responses": [ { "c_id": "1676", "date": "09 Nov 2015", "name": "Patrizio Tressoldi", "role": "Author Response", "response": "Thank you for your review. Here follows our replies to your comments.1. ......I would suggest to report previous studies of this type in the introduction and shortly report their findings (E.g. by Wackermann, Radin, Hinterberger,…).Reply: In the introduction and in the discussion of version 2, we added this information. Furthermore we added the list and references of all studies related to this line of investigation in the Supplementary Material.2. ....Spoken as a physicist, I do not really see the conditions for entanglement fulfilled because not every subsystem, that has information of another subsystem and shares the idea of interconnectedness behaves as being entangled. Therefore, it seems to be oversimplified to just state that \"this relationship constitutes an entangled state\".Reply: We are aware of your knowledge regarding the theory, and as explained by the GQT authors, this theory is not related to physical observables, but rather is one that can be applied to every complex system, regardless of whether physical, biological, or mental. If we assume that every human mind is a complex system, it is safe to assume the possibility that two minds, a certain distance apart, can be entangled if the intention to be connected is mutual, as suggested in the instructions to participants. This theory has already obtained empirical support in the entanglement of a human mind with a random number generator – considered to be a complex physical system (Walach et al. PLoS in press)....Another problem arises with the non-signal transfer paradigm which in this type of experiment would be violated if we would find replicable correlations of the same type.Reply: We agree. In the Discussion we clarify that what we observed is a correlation and not a transmission of signals.3. Why were you focusing on the frequency range between 9-10 Hz? Was this a post-hoc selection? Could you tell the results of the other standard frequencies such as theta, beta,.. bands?Reply: In the Discussion we specifically stated the explorative nature of the study. No other relevant results were observed in other EEG frequency bands.4-5. What is the GW6 method?Reply: As explained in the Data Analysis section, this is an alternative method for analyzing EEG signals based on the correlation among the signals detected in the EEG channels (14 in our case), which is more resistant to jitter and interferences compared to simple classical ERP averaging. The cited and freely available reference (Giroldini et al) contains a more technical explanation of how it works and how it differs from classical methods.6. Is it possible to tell something about the localization of your findings as you recorded 14 channels?Reply: We did not investigate the “sources” of the observed correlation given that our primary interest was in extracting it from the noise present in the EEG signals. We agree that a comparative analysis of the sources of the correlation, for example frontal versus occipital between the senders and the receivers, may give important clues about its characteristics.7. In the discussion please compare your findings to those of similar studies.Reply: Added." } ] }, { "id": "10191", "date": "29 Sep 2015", "name": "Aliodor Manolea", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe work is interesting and well done, in terms of the experimental work and data processing, both part of EEG signal processing and a statistical approach. The abstract describes very precisely the content of the article. The addressed research stage is clearly exposed and has direct addressability with the experiment described in this article and it is well supported by bibliographic references. The analysis method of ERP (The GW6 method), used to extract useful information from the noise, seemed to me very appropriate in the context of the uncertainty of ERP occurrence in the EEG recordings of the receivers. Also, statistical analysis is well designed and properly made, it allowed the exclusion of chance from investigated phenomenology. The effect size in such experiments is small or very small, being on the border of chance. Therefore, it would have been appropriate to make a comparison with the effect size obtained in other experiments of this type. The results are well supported by the experimental data and the processing methods. I think that the authors provide sufficient data to replicate the experiment without having great difficulties, which supports the scientific nature of the research. Also, I say that I agree with changes made at the suggestion of one of the referees. For future research I think that generating a baseline obtained from experimental sessions in which any (no) stimulus will be applied, It would be more appropriate than the method used in this experiment.", "responses": [ { "c_id": "1677", "date": "09 Nov 2015", "name": "Patrizio Tressoldi", "role": "Author Response", "response": "Thank you for your review. Here follows our replies to your comments.......5. The effect size in such experiments is small or very small, being on the border of chance. Therefore, it would have been appropriate to make a comparison with the effect size obtained in other experiments of this type.Reply: We are still wondering if a meta-analysis could be carried out on all the available studies related to this phenomena, but the variety of methods used to analyse the data (see the Supplementary Material), raise serious concerns on this possibility......8. For future research I think that generating a baseline obtained from experimental sessions in which any (no) stimulus will be applied, It would be more appropriate than the method used in this experiment.Reply: We agree with your suggestion we will take it in account for the next experiments." } ] } ]	1	https://f1000research.com/articles/4-457
https://f1000research.com/articles/4-666/v1	04 Sep 15	{ "type": "Correspondence", "title": "High Frequency Haplotypes are Expected Events, not Historical Figures", "authors": [ "Elsa G. Guillot", "Murray P. Cox", "Elsa G. Guillot" ], "abstract": "Cultural transmission of reproductive success states that successful men have more children and pass this raised fecundity to their offspring. Balaresque and colleagues found high frequency haplotypes in a Central Asian Y chromosome dataset, which they attribute to cultural transmission of reproductive success by prominent historical men, including Genghis Khan. Using coalescent simulation, we show that these high frequency haplotypes are consistent with a neutral model, where they commonly appear simply by chance. Hence, explanations invoking cultural transmission of reproductive success are statistically unnecessary.", "keywords": [ "Cultural Transmission of Reproductive Success", "Neutrality", "Haplotype Frequencies" ], "content": "\n\nCultural transmission of reproductive success states that successful men have more children and pass this raised fecundity on to their offspring. Observed in modern human populations from genealogies and surname studies1, in a genetic setting cultural transmission of reproductive success would cause particular male lines to dominate on the Y chromosome. Balaresque and colleagues2 examined a Y chromosome dataset from Central Asia to determine whether they could reconstruct historic instances of this behavior. Screening 8 microsatellites on the Y chromosome in 5,321 Central Asian men (distribution in Figure 1), they identified 15 haplotypes that are carried by more than 20 men (grey bars). The authors described these haplotypes as ‘unusually frequent,’ but did not provide any statistical support for this statement. These lineages were subsequently connected by the authors to prominent historical figures, including Genghis Khan and Giocangga.\n\nThe distribution (black and grey bars) is identical to Figure 2 of Balaresque et al2. Grey bars indicate the 15 haplotypes that Balaresque and colleagues describe as ‘unusually frequent.’ Red shading indicates the 95% confidence intervals of haplotype frequencies from one million simulations under a fitted neutral model. All of the high frequency haplotypes (grey bars) fall within these 95% confidence bounds.\n\nHowever, in any given haplotype frequency distribution, a number of haplotypes are expected to occur at high frequency simply by chance. In neutrally evolving systems, haplotype frequency distributions follow a Zipfian power law3: most lineages are carried by only a few men (Figure 1, left side), while a small number of lineages are carried by many men (Figure 1, right side). The Y chromosome distribution observed by Balaresque and colleagues closely follows such a power law, thus providing strong preliminary evidence that their Y chromosome dataset may be selectively neutral.\n\nTo more explicitly test whether the observed high frequency haplotypes are actually unusually frequent, we simulated genetic data under the standard coalescent, a neutral model that does not include cultural transmission of reproductive success. We modeled the evolution of 5,321 Y chromosomes, each carrying 8 fully linked microsatellites, to match the observed data. The code for these simulations, including full details of parameter values, is available online (http://elzaguillot.github.io/Allele-Frequency-Spectrum-simulations).\n\nSimulations were first run across a sweep of θ values to find the best match with the power law distribution observed in the Central Asian Y chromosome dataset. The least squares fit between observed and simulated distributions was minimized at θ = 131. In one million simulations run at this value, we found that 27.2% of the simulations contained at least 15 haplotypes carried by more than 20 men, thus illustrating that high frequency haplotypes like those observed among Central Asian Y chromosomes are relatively common, even when cultural transmission of reproductive success is not acting. The Y chromosome haplotype frequency distribution observed by Balaresque and colleagues falls within the 95% confidence intervals of our simulations (Figure 1, red shading).\n\nThe most parsimonious explanation is therefore that the high frequency haplotypes observed by Balaresque and colleagues in Central Asia are simply expected chance events. While we strongly encourage further research into cultural transmission of reproductive success, no statistical evidence has been presented to show that this process has acted on this particular dataset of Central Asian Y chromosomes. As no additional evidence is presented in support of proposed links to famous historical men, these haplotypes instead most likely reflect the chance proliferation of random male lines, probably from historically unrecorded, culturally undistinguished, but biologically lucky Central Asian men.\n\n\nSoftware availability\n\nhttp://elzaguillot.github.io/Allele-Frequency-Spectrum-simulations\n\nhttp://dx.doi.org/10.5281/zenodo.298884\n\nLesser GNU Public License 3.0 https://www.gnu.org/licenses/lgpl.html", "appendix": "Author contributions\n\n\n\nEGG conceived the study and carried out the research. EGG and MPC designed the experiments and wrote the manuscript. Both 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\nReferences\n\nKolk M: Multigenerational transmission of family size in contemporary Sweden. Popul Stud (Camb). 2014; 68(1): 111–129. PubMed Abstract \| Publisher Full Text\n\nBalaresque P, Poulet N, Cussat-Blanc S, et al.: Y-chromosome descent clusters and male differential reproductive success: young lineage expansions dominate Asian pastoral nomadic populations. Eur J Hum Genet. 2015; In Press. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBerestycki J, Berestycki N, Limic V: Asymptotic sampling formulae for Λ-coalescents. Ann I H Poincaré-Pr. 2014; 50(3): 715–731. Publisher Full Text\n\nGuillot EG, Cox MP: Allele Frequency Spectrum simulations. Zenodo. 2015. Data Source" }	[ { "id": "10223", "date": "01 Oct 2015", "name": "Sohini Ramachandran", "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\nGuillot and Cox present a very interesting criticism of Balaresque et al.'s work in press on high frequency haplotypes in Central Asian Y chromosomes, by showing that distributions like those observed by Balaresque and colleagues can be observed using neutral simulations.I have three comments I would like to see the authors address:The \"Genghis Khan reproductive success\" hypothesis emerged in Zerjal et al.'s work in 2003 and I think it would be helpful for the authors to comment on what analyses in that work support Zerjal et al.'s conclusions; can their simulations reproduce what Zerjal et al. observed under strictly neutral processes without a high number of mergers in the coalescent process?The phrase \"historically unrecorded, culturally undistinguished, but biologically lucky Central Asian men.\" should be changed to \"historically unrecorded but biologically lucky Central Asian men.\"The authors should provide sample output files for their simulation pipeline for users to analyze, and their code so that the number of simulations run is a user-provided argument. Given that the pipeline can take at least hours and perhaps days to generate the million simulations they studied, a toy example is worth looking at quickly and the authors could allow readers to generate examples more quickly without needing to fiddle with the bash/python/R pipeline on their own.", "responses": [ { "c_id": "1782", "date": "09 Feb 2016", "name": "Murray Cox", "role": "Reader Comment", "response": "We thank Sohini Ramachandran for her careful review and address her three comments below.As noted by this reviewer, the 2015 paper by Balaresque and colleagues builds on earlier work from 2003 that first identified a Y chromosome lineage at high frequency in Central Asia and putatively linked it to Genghis Khan (Zerjal T, Xue Y, Bertorelle G, et al.: The genetic legacy of the Mongols. Am J Hum Genet. 2003; 72:717-721 http://doi.org/10.1086/367774). Just as the 2015 paper provided no statistical support for the association reported in that work, none is provided in the 2003 paper either. We were particularly taken by the idea of running the analysis presented here on the original 2003 data set, but unfortunately, the 2003 data were not released either as supplementary material to the original paper or uploaded to a public repository. One of us (EG) contacted the lead author (Tatiana Zerjal, now based at INRA, France), who could not provide the original data set. A re-analysis, along the lines of that presented in our report, is therefore not possible.We agree. We argue that the data are consistent with lineages reaching high frequency simply by chance, but make no claims about the cultural and/or historical roles of these (random) ancestors. We agree. Intermediate files have now been added to the Git repository, which will allow readers to explore our statistical re-analysis without needing to run the entire pipeline. Note, however, that we would strongly encourage researchers who wish to critique this work to repeat our analyses in full." } ] }, { "id": "11823", "date": "15 Jan 2016", "name": "Heather Norton", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummaryIn this manuscript Guillot and Cox test the claim made by Balaresque et al. (2015) that a subset of Y-chromosome haplotypes from Central Asian men occur at “unusually” high frequency, possibly indicating social selection for men carrying these lineages. Using simulations designed to match the data reported by Balaresque et al. the authors demonstrate that the reported distribution of Y chromosome haplotypes can be obtained under neutral conditions. This suggests that it is not necessary to invoke a model that includes cultural transmission of reproductive success to explain the observed distribution. CommentWhile the focus of this correspondence article is on the Balaresque data, can the authors briefly comment on other papers that have also investigated cultural transmission of reproductive success—specifically, have there been other studies that report high frequency Y haplotypes in other populations that are not consistent with neutrality?", "responses": [ { "c_id": "1783", "date": "09 Feb 2016", "name": "Murray Cox", "role": "Reader Comment", "response": "We thank Heather Norton for her careful review and address her comment below.Cultural transmission of reproductive success has been identified from historic (i.e., non-genetic) records, with at least one instance in Quebec linked to higher prevalence of genetic disorders (Austerlitz F, Heyer E: Social transmission of reproductive behavior increases frequency of inherited disorders in a young-expanding population. Proc Natl Acad Sci USA. 1998; 95:15140-15144). Cultural transmission of reproductive success has also been shown to leave identifiable signals in genetic data, and for haploid loci, these signals seem to be most clear from tree imbalance metrics (Heyer E. Sibert A, Austerlitz F: Cultural transmission of fitness: genes take the fast lane. Trends Genet. 2005; 21:234-239. http://doi.org/10.1016/j.tig.2005.02.007). We therefore believe that cultural transmission of reproductive success has likely acted in some human populations. Indeed, we have observed rare deviations from neutral expectations in our own work (Lansing J, Watkins J, Hallmark B et al.: Male dominance rarely skews the frequency distribution of Y chromosome haplotypes in human populations. Proc Natl Acad Sci USA. 2008; 105:11645-11650. http://doi.org/10.1073/pnas.0710158105).However, the association commonly made between a high frequency lineage in Central Asia and Genghis Khan is easily the most well known claim for cultural transmission of reproductive success in humans. Our aim here is to show that statistical support for this particular assertion is currently lacking." } ] }, { "id": "11952", "date": "18 Jan 2016", "name": "Nick Patterson", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis short note considers the recent paper by Balaresque et al.1 on the distribution of Y-chromosome haplotypes in Central Asia. Through simulation they show that the haplotype frequency distribution is not very surprising and suggest that the results of1 are most likely due to chance.But there is more to the analysis of1 than just the haplotype frequency. Their analysis groups haplotypes into `descent clusters', estimates the time to the most recent common ancestor (TMRCA) and looks into the spatial distribution of the haplotypes. None of this was simulated. There is no formal test applied in1, but visually the results look to this reviewer very surprising under a scenario where to quote Guillot and Cox the results arechance proliferation of random male lines... from culturally undistinguished but biologically lucky...menIn autosomal analysis of admixture events2, overwhelming genetic evidence was found for the Mongol expansion across Eurasia. This by no means shows that the Y-chromosome signal was, at least partially, driven by high status Mongols, but to this reviewer this still seems more likely than not.", "responses": [ { "c_id": "1784", "date": "09 Feb 2016", "name": "Murray Cox", "role": "Reader Comment", "response": "We thank Nick Patterson for his careful review and address his comments below.We did not focus on population structure for two reasons. First, our primary aim was simply to raise awareness that the original paper did not perform any statistical testing to show that its high frequency lineages were actually ‘unusually frequent.’ Under a simple neutral model, we show that they are not. Second, and perhaps more importantly, the population structure of the study system was not described in sufficient detail for us to implement a formal structured demographic model. Such testing is an obvious next step and we would be very interested to see the results, but we also suggest that these tests should really be run by the original authors.However, the reviewer notes two particular features of population structure that require more detailed discussion: the spatial clustering of the high frequency haplotypes, and their young ages.Imagine that we can perfectly track the history of a neutral, but high frequency, haplotype through time. Initially, the haplotype arises by mutation within a single population. Over time, the haplotype increases in frequency, and via migration, spreads to neighboring demes (whether defined geographically or culturally). Even in a neutral setting, high frequency haplotypes will therefore appear to be clustered.Assuming a high frequency haplotype is not lost (or fixed) through drift, it will also experience mutation over time, which will change instances of the high frequency haplotype into new derived forms. Eventually, the original haplotype will be lost entirely in this way, replaced by derived descendants. Consequently, when high frequency haplotypes are observed in a neutral setting, they are likely to be disproportionately young. High frequency neutral lineages can only persist for a short time before their frequency is reduced again through mutation or drift (unless, of course, they become fixed).The high frequency haplotypes identified by Balaresque and colleagues are spatially structured and young, but neither pattern is diagnostic of cultural transmission of reproductive success. As shown by the thought experiment above, these two features can equally be expected under a purely neutral scenario.We agree completely with the reviewer that the Mongol expansion heavily impacted the genetic profile of Central Asia, and like him, accept that many of high frequency Y chromosome lineages may trace back to members of the Mongol army. Our main point, though, is that no statistical evidence has yet been presented to show that these lineages reached high frequency due to cultural transmission of reproductive success. Nor is there any evidence that they trace back to historically important men like Genghis Khan, rather than any other random men in his vast Mongol army." } ] } ]	1	https://f1000research.com/articles/4-666
https://f1000research.com/articles/5-153/v1	09 Feb 16	{ "type": "Review", "title": "Evaluation and management of anterior urethral stricture disease", "authors": [ "Altaf Mangera", "Nadir Osman", "Christopher R. Chapple", "Nadir Osman", "Christopher R. Chapple" ], "abstract": "Urethral stricture disease affects many men worldwide. Traditionally, the investigation of choice has been urethrography and the management of choice has been urethrotomy/dilatation. In this review, we discuss the evidence behind the use of ultrasonography in stricture assessment. We also discuss the factors a surgeon should consider when deciding the management options with each individual patient. Not all strictures are identical and surgeons should appreciate the poor long-term results of urethrotomy/dilatation for strictures longer than 2 cm, strictures in the penile urethra, recurrent strictures, and strictures secondary to lichen sclerosus. These patients may benefit from primary urethroplasty if they have many adverse features or secondary urethroplasty after the first recurrence.", "keywords": [ "Urethral stricture disease", "urethrotomy", "dilatation", "urethrography" ], "content": "Introduction\n\nThis update will concentrate on the advances in the investigation and management of urethral stricture disease in men. Traditionally, urologists have offered such men urethral dilatation/urethrotomy, which carries the risk of needing repeat interventions and a long-term need to self-dilate the urethra on a regular basis. We aim to discuss the evidence relating to patient investigation and selection for more definitive surgery such as urethroplasty.\n\nA urethral stricture is a narrowing of the urethra. A “true” stricture is the result of ischaemic spongiofibrosis manifesting as scar tissue in the corpus spongiosum1. Contraction of this scar tissue leads to a reduction in the urethral calibre, which leads to voiding difficulty. On the other hand, urethral distraction injuries occur as a result of blunt trauma distracting the two ends of the urethra apart and are not “true” strictures. Ischaemic spongiofibrosis may be due to infection such as gonococcal urethritis, inflammation such as lichen sclerosus, or instrumentation; however, the majority of strictures are idiopathic. It is reported that, in the US, on the basis of 10 public and private databases between 1992 and 2000, there were 5 million office visits per year and more than 5,000 inpatient admissions per year due to urethral strictures2.\n\nThere are many management options available for treating urethral stricture disease, commencing with less invasive urethral dilatation, urethral stenting and urethrotomy, and progressing to anastomotic and augmentation urethroplasty. The optimum management approach is often debated by urologists; some prefer a less invasive approach and perform urethrotomy/dilatation as first and even second line for all patients, whereas those who perform urethroplasty regularly recognise that urethroplasty may become more difficult after urethrotomy and advocate primary urethroplasty, as it is thought that a urethrotomy lengthens the stricture and leads to deepening of the spongiofibrosis, resulting in poorer blood supply to the urethra. Also, in a multivariate analysis of urethroplasty outcomes, prior urethrotomy was found to be a risk factor for failure3. Others take a “middle of the road” approach and select patients in whom urethrotomy is likely to have a limited role and counsel them regarding primary urethroplasty. In this article, we aim to review the literature regarding the investigation and selection of patients for urethroplasty.\n\n\nDiscussion\n\nMuch work has gone into providing a means of identifying the extent of a urethral stricture pre-operatively. Traditionally, a retrograde urethrogram is used to identify stricture density and length. Ideally, an antegrade and retrograde urethrogram could be performed to fully characterise the stricture. A recent study has suggested that the operating urologist may be better off performing and interpreting the urethrogram, as this led to the most accurate finding of strictures and description of stricture length4. In this study, all urethrograms were performed by a urologist and therefore it is not known whether the radiologist would have obtained the same results had they performed and reported the studies. Certainly, in our practice, a sub-speciality uro-radiologist performs and reports the study and it is thereafter viewed by the operating surgeon.\n\nAnother method of stricture assessment involves the use of ultrasound. This can accurately assess the extent of ischaemic spongiofibrosis in the corpus spongiosum and this is often longer than the “white” stricture which is seen at endoscopy, which in turn is longer than the “narrowing” seen on the urethrogram5. A recent report from McAninch et al. has shown that this can pick up underlying spongiofibrosis, which changed the stricture length in 45% of patients over a urethrogram6. In this series of 232 men, the urethroplasty approach was changed in 19% of patients. Strictures which appear short on urethrography but have extensive underlying spongiofibrosis are important to identify as these are more likely to require substitution urethroplasty instead of anastomotic urethroplasty. In the series from McAninch et al., the mean stricture length was increased by the use of ultrasonography from 2 to 3.4 cm. Another study, of 40 patients, has suggested that ultrasound is accurate in assessing anterior urethral strictures and also provides more information than the urethrogram alone7. Ultrasound has also been found to be equivalent to magnetic resonance urethrography and the latter is probably unnecessary for assessing the anterior urethra8.\n\nAnother important use for ultrasound, which has not been investigated, is in deciding which patients are likely to benefit from a urethral dilatation/urethrotomy or will likely require a urethroplasty. It is thought that by incising a stricture, the underlying fibrosis is lengthened and thus subsequent stricturing, if it recurs, is likely to lead to a longer stricture. In an interesting retrospective study, it was shown that bulbar strictures where urethrotomy/dilatation had been undertaken two or more times were longer and also recurred quicker than in those who underwent one or no transurethral surgery9. The difficulty here is that we have “the cause or effect scenario” as it cannot be proven, in this study, whether the strictures undergoing more transurethral surgery were longer and therefore more apparent symptomatically to begin with.\n\nThe capability of a urethra to heal without re-stricturing is reliant upon an adequate underlying blood supply and therefore knowledge of underlying spongiofibrosis may well be beneficial in identifying those patients who may benefit from primary urethroplasty. A recent Société Internationale d’Urologie/International Consultation on Urological Diseases (SIU/ICUD) consultation for evaluation and follow-up of urethral stricture disease concluded that urethrography and urethroscopy remain the investigations of choice for the anterior urethra10. The evidence for the use of ultrasound is currently limited but does show some promise in evaluating underlying spongiofibrosis and may help counsel patients better before undertaking surgery as to the type of surgery they may require.\n\nTraditionally, the most commonly performed procedure for urethral stricture has been dilation/urethrotomy. A survey of 1,262 American urologists found that most urologists treat between 6 and 20 strictures per year and over 90% performed dilatation/urethrotomy11. It is noteworthy that 74% of urologists believed that urethroplasty should be performed only after repeated failure of endoscopic methods.\n\nA randomised controlled trial reported by Steenkamp et al. reviewed 210 men, of whom 106 underwent dilatation and 104 urethrotomy12. At 1 year, there was a success rate of 60% if the stricture was less than 2 cm, 50% if it was between 2 and 4 cm, and 20% if it was more than 4 cm in length. In a subsequent publication, Heyns et al., working on the same dataset, looked at repeat dilatation/urethrotomy and noted that after a single treatment 70% would be stricture-free at 3 months, 35–40% would remain stricture-free at 48 months, and a secondary procedure was of limited benefit at 24 months, but not at 48 months13. A third treatment was of no benefit at all. Other authors have reported even worse success rates, with primary urethrotomy approaching even less than 40%14,15. However, these are retrospective case reports and caution should be applied when interpreting their findings.\n\nGreenwell et al. concluded that, in terms of cost-effectiveness, the use of urethroplasty after failure of one urethrotomy/dilatation was likely to be the most cost-effective approach16. Contrastingly, Rourke and Jordan have suggested that treatment of short bulbar strictures by urethroplasty is more cost-effective than urethrotomy17. A calculated approach is that initial urethrotomy followed by urethroplasty is the most cost-effective approach if there is recurrence of the stricture, unless the success rate of urethrotomy was likely to be inferior to 35%18. Certainly, experts who perform urethroplasty regularly do feel it is made more difficult by repeated interventions such as urethrotomy. A retrospective review by Roehrborn and McConnell reported doubling of the failure rate in patients with previous surgical manipulation19. Similarly, Breyer et al. reported a hazard ratio of 1.7 on multivariate analysis of 443 patients if they had previously undergone urethrotomy3.\n\nThe reasons some urologists offer repeat urethrotomy are manifold. A recent study of case logs from the US showed great disparity in the number of urethroplasties performed in different regions, and men were more likely to be referred for specialist intervention by newly certified urologists than established urologists20. All urologists are familiar with urethrotomy/dilatation and therefore are more likely to offer this than refer to another institute where urethroplasty is performed regularly. In addition, patient comorbidities may exclude a patient from having urethroplasty21. Similar findings were reported by a European study, in which 79% of Dutch urologists reported that they felt a urethroplasty should be offered only after failed urethrotomy15. The authors found that 20% of urologists would continue to perform urethrotomy for a 1 cm stricture even after two recurrences. Therefore, it transpires for some patients that urethroplasty is not considered at all or considered only when the stricture is lengthy and subsequent urethroplasty more difficult.\n\nTable 1 lists the factors which should be considered as risk factors for recurrence of urethral strictures22. In our experience, bulbar strictures are likely to recur less often after urethrotomy than penile urethral strictures because of better blood supply in the bulbar urethra, although there are limited data on this in the literature. Strictures which recur after urethrotomy/dilatation are almost certainly likely to need further intervention after a repeat urethrotomy/dilatation. We feel that by assessing these in each individual patient, three categories of patients can be created. Those with multiple risk factors should proceed to urethroplasty if suitable, and those with two risk factors may undergo primary urethrotomy/dilatation but should be counselled regarding urethroplasty. Finally, those with only one risk factor could undergo urethrotomy/dilatation first. This approach, though useful in clinical practice, does require some fine tuning, and only with emerging evidence will it be possible to give a weighting to each risk factor.\n\nMany surgeons will rely upon the visual appearance of the urethra at cystourethroscopy; an ischaemic urethra looks white or grey, and healthy well-vascularised tissue appears pink. The narrowed portion of the urethra may appear much shorter than the white area with underlying spongiofibrosis. Cystourethroscopy provides earlier evidence of stricturing or recurrence usually prior to a reduction in flow rate23. The flow rate is not significantly affected until the urethral calibre is less than 11 Fr24. In our experience, we advocate symptom assessment and cystourethroscopy for follow-up. The frequency and length of follow-up should follow a risk-stratified approach as discussed above for patient assessment (Table 1). The evidence for this approach, however, is currently lacking in the literature.\n\nPatients with strictures may also present with a multitude of symptoms which may or may not impact upon their quality of life25. In this regard, a Patient-Reported Outcome Measure (PROM) has been validated for this cohort26. This also takes account of sexual function and should be used in the assessment of patients pre- and post-operatively. A more recent report has shown this PROM to be able to detect post-operative changes after 2 years of follow-up27.\n\nQuestions remaining for the future include the use of ultrasound in deciding which options are beneficial for patients prior to the first urethrotomy or for urethroplasty. Further investigation is required into factors that lead to lower success with urethrotomy, which are also those leading to worse outcomes with urethroplasty. The results of the Open urethroplasty versus Endoscopic urethrotomy (OPEN) randomised controlled trial are eagerly awaited. This specifically looks at the use of urethroplasty or urethrotomy for recurrent strictures. A Cochrane review has shown the dearth of randomised controlled data in this field and these are what we really require28. Depending on the findings of the OPEN trial, the field should be able to progress in the correct direction and lead to better patient selection and, ultimately, improved patient outcomes.\n\n\nConclusions\n\nFrom the discussion above, it is clear that proper assessment of urethral strictures is required to dictate appropriate management. All too often, a stricture is seen via flexible cystoscopy and the patient placed on the waiting list for a urethrotomy which is left to a junior resident to “cut through”. Surgeons should appreciate that by cutting through a stricture they may be lengthening the stricture as it relies on an adequate residual blood supply to heal without scarring. It should be recognised that the success of their intervention is limited by certain stricture characteristics and not all strictures are the same. Variations in strictures should be noted and respected. Stricture length, location and aetiology have been shown to affect recurrence rate, and urologists should be able to classify strictures as high-, intermediate- and low-risk (Table 1). With these factors in mind, a urologist should be more equipped in counselling patients regarding their options for management and follow-up.\n\n\nAbbreviations\n\nOPEN, Open urethroplasty versus Endoscopic urethrotomy; PROM, Patient-Reported Outcome Measure.", "appendix": "Competing interests\n\n\n\nThe authors declare that they 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\nMundy AR: Urethral surgery. In R Turner Warwick, ed. Current operative surgery: urology. Chapt 12. 1988; 160–218.\n\nSantucci RA, Joyce GF, Wise M: Male urethral stricture disease. J Urol. 2007; 177(5): 1667–74. PubMed Abstract \| Publisher Full Text\n\nBreyer BN, McAninch JW, Whitson JM, et al.: Multivariate analysis of risk factors for long-term urethroplasty outcome. J Urol. 2010; 183(2): 613–7. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nBach P, Rourke K: Independently interpreted retrograde urethrography does not accurately diagnose and stage anterior urethral stricture: the importance of urologist-performed urethrography. Urology. 2014; 83(5): 1190–3. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nBarbagli G, Azzaro F, Menchi I, et al.: Bacteriologic, histologic and ultrasonographic findings in strictures recurring after urethrotomy. A preliminary study. Scand J Urol Nephrol. 1995; 29(2): 193–5. PubMed Abstract \| Publisher Full Text\n\nBuckley JC, Wu AK, McAninch JW: Impact of urethral ultrasonography on decision-making in anterior urethroplasty. BJU Int. 2012; 109(3): 438–42. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nRavikumar BR, Tejus C, Madappa KM, et al.: A comparative study of ascending urethrogram and sono-urethrogram in the evaluation of stricture urethra. Int Braz J Urol. 2015; 41(2): 388–92. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nEl-Ghar MA, Osman Y, Elbaz E, et al.: MR urethrogram versus combined retrograde urethrogram and sonourethrography in diagnosis of urethral stricture. Eur J Radiol. 2010; 74(3): e193–8. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nHudak SJ, Atkinson TH, Morey AF: Repeat transurethral manipulation of bulbar urethral strictures is associated with increased stricture complexity and prolonged disease duration. J Urol. 2012; 187(5): 1691–5. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nAngermeier KW, Rourke KF, Dubey D, et al.: SIU/ICUD Consultation on Urethral Strictures: Evaluation and follow-up. Urology. 2014; 83(3 Suppl): S8–17. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nBullock TL, Brandes SB: Adult anterior urethral strictures: a national practice patterns survey of board certified urologists in the United States. J Urol. 2007; 177(2): 685–90. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nSteenkamp JW, Heyns CF, de Kock ML: Internal urethrotomy versus dilation as treatment for male urethral strictures: a prospective, randomized comparison. J Urol. 1997; 157(1): 98–101. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nHeyns CF, Steenkamp JW, De Kock ML, et al.: Treatment of male urethral strictures: is repeated dilation or internal urethrotomy useful? J Urol. 1998; 160(2): 356–8. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nPansadoro V, Emiliozzi P: Internal urethrotomy in the management of anterior urethral strictures: long-term followup. J Urol. 1996; 156(1): 73–5. PubMed Abstract \| Publisher Full Text\n\nvan Leeuwen MA, Brandenburg JJ, Kok ET, et al.: Management of adult anterior urethral stricture disease: nationwide survey among urologists in the Netherlands. Eur Urol. 2011; 60(1): 159–66. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nGreenwell TJ, Castle C, Andrich DE, et al.: Repeat urethrotomy and dilation for the treatment of urethral stricture are neither clinically effective nor cost-effective. J Urol. 2004; 172(1): 275–7. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nRourke KF, Jordan GH: Primary urethral reconstruction: the cost minimized approach to the bulbous urethral stricture. J Urol. 2005; 173(4): 1206–10. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nWright JL, Wessells H, Nathens AB, et al.: What is the most cost-effective treatment for 1 to 2-cm bulbar urethral strictures: societal approach using decision analysis. Urology. 2006; 67(5): 889–93. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nRoehrborn CG, McConnell JD: Analysis of factors contributing to success or failure of 1-stage urethroplasty for urethral stricture disease. J Urol. 1994; 151(4): 869–74. PubMed Abstract \| F1000 Recommendation\n\nBurks FN, Salmon SA, Smith AC, et al.: Urethroplasty: a geographic disparity in care. J Urol. 2012; 187(6): 2124–7. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nHeyns CF, van der Merwe J, Basson J, et al.: Treatment of male urethral strictures - possible reasons for the use of repeated dilatation or internal urethrotomy rather than urethroplasty. S Afr J Surg. 2012; 50(3): 82–7. PubMed Abstract \| F1000 Recommendation\n\nKinnaird AS, Levine MA, Ambati D, et al.: Stricture length and etiology as preoperative independent predictors of recurrence after urethroplasty: A multivariate analysis of 604 urethroplasties. Can Urol Assoc J. 2014; 8(5–6): E296–300. PubMed Abstract \| Publisher Full Text\n\nGoonesinghe SK, Hillary CJ, Nicholson TR, et al.: Flexible cystourethroscopy in the follow-up of posturethroplasty patients and characterisation of recurrences. Eur Urol. 2015; 68(3): 523–9. PubMed Abstract \| Publisher Full Text\n\nSmith JC: The measurement and significance of the urinary flow rate. Br J Urol. 1966; 38(6): 701–6. PubMed Abstract \| Publisher Full Text\n\nRourke K, Hickle J: The clinical spectrum of the presenting signs and symptoms of anterior urethral stricture: detailed analysis of a single institutional cohort. Urology. 2012; 79(5): 1163–7. PubMed Abstract \| Publisher Full Text\n\nJackson MJ, Sciberras J, Mangera A, et al.: Defining a patient-reported outcome measure for urethral stricture surgery. Eur Urol. 2011; 60(1): 60–8. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nJackson MJ, Chaudhury I, Mangera A, et al.: A prospective patient-centred evaluation of urethroplasty for anterior urethral stricture using a validated patient-reported outcome measure. Eur Urol. 2013; 64(5): 777–82. PubMed Abstract \| Publisher Full Text\n\nWong SS, Aboumarzouk OM, Narahari R, et al.: Simple urethral dilatation, endoscopic urethrotomy, and urethroplasty for urethral stricture disease in adult men. Cochrane Database Syst Rev. 2012; 12: CD006934. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation" }	[ { "id": "12336", "date": "09 Feb 2016", "name": "Richard A Santucci", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12337", "date": "09 Feb 2016", "name": "Keith F Rourke", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-153
https://f1000research.com/articles/5-152/v1	09 Feb 16	{ "type": "Review", "title": "Recent Advances in Understanding and Managing Tourette Syndrome", "authors": [ "Mary Ann Thenganatt", "Joseph Jankovic", "Mary Ann Thenganatt" ], "abstract": "Tourette syndrome (TS) is a neurologic and behavioral disorder consisting of motor and phonic tics with onset in childhood or adolescence. The severity of tics can range from barely perceptible to severely impairing due to social embarrassment, discomfort, self-injury, and interference with daily functioning and school or work performance. In addition to tics, most patients with TS have a variety of behavioral comorbidities, including attention deficit hyperactivity disorder and obsessive-compulsive disorder. Studies evaluating the pathophysiology of tics have pointed towards dysfunction of the cortico-striato-thalamo-cortical circuit, but the mechanism of this hyperkinetic movement disorder is not well understood. Treatment of TS is multidisciplinary, typically involving behavioral therapy, oral medications, and botulinum toxin injections. Deep brain stimulation may be considered for “malignant” TS that is refractory to conventional therapy. In this review, we will highlight recent developments in the understanding and management strategies of TS.", "keywords": [ "Tourette syndrome", "neurology", "behaviour" ], "content": "Introduction\n\nTourette syndrome (TS) is a neurobehavioral disorder characterized by motor and phonic tics that begin in childhood or adolescence. Adult-onset tics usually represent recurrences of childhood tics, although there are some tic disorders, other than TS, that can initially manifest in adulthood1. A tic is an abrupt and brief movement or sound that is often preceded by a local or generalized urge or some other premonitory sensation. Thus, tic is an example of a group of hyperkinetic movement and other motor disorders that are increasingly recognized to be associated with a sensory phenomenon2. Tics can be classified as clonic (jerk-like), dystonic (sustained), tonic (isometric), and blocking (cessation of movement and speech). In addition to its brief, intermittent, and repetitive nature, other features of tics include suggestibility, suppressibility, and distractibility, which may lead to a wrong diagnosis of a tic as a “psychogenic” movement disorder3–5. There is no diagnostic test for TS, but the Diagnostic and Statistical Manual of Mental Disorders Fifth Edition’s definition of TS requires the presence of multiple motor and phonic tics with onset prior to age 18 and lasting at least 1 year6.\n\nThe epidemiologic findings in TS vary depending on the population studied, methodology, and many other factors. Among 21 population-based prevalence studies, the pooled TS population prevalence was estimated to be 0.52% (95% confidence interval [CI]: 0.32–0.85)7. In one study, based on cross-sectional structured diagnostic interviews in 1374 TS patients and 1142 TS-unaffected family members, the lifetime prevalence of comorbid disorders was 85.7%; 57.7% of the population had two or more psychiatric disorders8. Furthermore, 72.1% of the individuals met the criteria for obsessive-compulsive disorder (OCD) or attention deficit/hyperactivity disorder (ADHD), and of other disorders, including mood, anxiety, and disruptive behavior, each was present in approximately 30% of the patients. The age at greatest risk for the onset of most comorbid psychiatric disorders was between 4 and 10 years of age. The median age of onset of TS was 6 years of age.\n\nIn this review, we discuss the most recent developments in our understanding of the underlying pathophysiology and pathogenesis of TS as well as management and novel therapeutic strategies.\n\n\nPathophysiology\n\nVarious physiologic, imaging, and genetic techniques have been used to provide insights into the pathophysiology of TS. TS has been considered a developmental disorder, but evidence for this hypothesis has been lacking9,10. Using 3-Tesla structural neuroimaging, one study examined cortical thickness in 52 TS patients and matched controls and found reduced depth and thickness of the gray matter in the pre- and post-central and superior, inferior, and internal frontal sulci, which correlated with tic severity11. Abnormal brain plasticity involving the motor cortex, basal ganglia, and brainstem has been suggested as a mechanism for altered motor control processing in TS12,13.\n\nFunctional imaging and animal studies have pointed to TS as a network disorder with dysfunction in the cortico-striato-thalamo-cortical circuit9,14,15. For example, one functional magnetic resonance imaging (fMRI) study evaluated 36 adult TS patients and found that temporal progression of structures involved during the generation of a tic followed a sequence of activation in the cortico-striato-thalamo-cortical circuit16. Thus, 1–2 seconds before a tic, the supplementary motor area (SMA), ventral primary motor cortex, primary sensorimotor cortex, and parietal operculum exhibited activation, followed by the anterior cingulate, putamen, insula, amygdala, cerebellum, and the extrastriatal-visual cortex. During the onset of the tic, the thalamus, central operculum, and primary motor and somatosensory cortices became activated. Multimodal imaging techniques in 16 TS patients found that functional connectivity between anterior insular and sensorimotor cortex was increased17. Diffusion tensor imaging techniques have shown reduced connectivity of frontal brain networks involved in planning and controlling actions in TS patients18.\n\nThe cause of the disruption of the cortico-basal ganglia structures is not fully understood. Abnormalities of the γ-aminobutyric acid (GABA) system have been proposed as contributing to the disinhibition seen in TS. The use of positron emission tomography and [11C]flumazenil, a GABA receptor ligand, has identified abnormalities in the GABAergic system in TS patients, including decreased GABA-A receptor binding in the ventral striatum, globus pallidus, thalamus, amygdala, and right insula and increased binding in the bilateral substantia nigra, left periaqueductal grey, right posterior cingulate cortex, and bilateral cerebellum19.\n\nA major unmet need in TS research is clinical-pathological correlation. One neuropathological study investigated the density of interneurons and medium spiny neurons in the striatum of five TS brains compared to normal controls20. In TS brains, there was a 50–60% reduction in cholinergic and parvalbumin interneurons, the latter representing inhibitory GABAergic interneurons in the striatum.\n\nStudies of the urge, or premonitory sensation, associated with tics have identified neural networks associated with these sensory phenomena2. Abnormalities in the sensorimotor cortex and somatosensory cortex have been found to be associated with the premonitory sensation9. Imaging studies have suggested that areas outside of the sensorimotor network, such as the insula, also contribute to this urge. In one study using resting-state fMRI in 13 TS patients and 13 controls, the investigators found increased connectivity between the right dorsal anterior insula and the frontal-striatal nodes of the urge-tic network and the bilateral SMA which correlated with urge severity, thus suggesting that the anterior insula is involved in interoceptive awareness of sensations leading to the urge to tic21.\n\n\nGenetics\n\nWhile TS is clearly a familiar disorder, often with bilineal inheritance (both parents affected)22, our understanding of the genetic underpinnings of TS is still in its infancy23. A population-based study in Sweden found overall heritability of tic disorders to be 0.77, increasing with the degree of genetic relatedness: odds ratio (OR) of first-degree relatives (18.69) greater than that of second degree relatives (OR 4.58), which was greater than that of third-degree relatives (OR 3.07)24. Despite the strong heritability of TS, a causative gene or genes have yet to be discovered25. Various study designs have evaluated the genetics of TS including twin studies, linkage analyses, cytogenic abnormalities, copy number variation studies, and genome-wide association studies (GWAS)23. Studies of multigenerational pedigrees of TS families have found an association between a functional mutation in the histamine decarboxylase (HDC) gene and TS26,27. The HDC gene encodes for L-histidine decarboxylase, which is the rate-limiting step enzyme in histamine production. The first GWAS in TS included 1285 cases and 4964 controls of European ancestry28. In the primary analysis, no markers achieved a genome-wide threshold of significance (p<5×10-8); the strongest signal was found in rs7868992 on chromosome 9q32 within COL27A1 (p=1.85×10-6). In a subsequent study, 42 single nucleotide polymorphisms (SNPs) (p<10-3) from this GWAS were genotyped in 609 additional cases and 610 controls29. One SNP, rs2060546, was significantly associated with TS in this sample (p=3.3×10-4, OR=2.41) and when analyzed with the original GWAS data, this association was even greater (p=5.8×10-7, OR=1.77). This SNP, rs2060546, is located on chromosome 12q22 close to NTN4, a gene that codes for a protein in the developing striatum involved in axon guidance and outgrowth. In an attempt to examine the contribution of large, rare copy number variants to TS and OCD susceptibility, a genome-wide, cross-disorder study in 2699 cases (1086 TS, 1613 OCD) and 1789 controls found deletions in 16p13.1130. This suggests that mutations in this region may be associated with increased susceptibility to TS, OCD, and possibly other neurodevelopmental disorders. Based on a GWAS in 2723 cases (1310 with OCD, 834 with TS, and 579 with OCD plus TS), 5667 ancestry-matched controls, and 290 OCD parent-child trios, no individual SNPs achieved genome-wide significance31. However, polygenic score analyses identified a significant polygenic component for OCD but not for TS. The study concluded that “OCD with co-occurring TS may have different underlying genetic susceptibility compared with OCD alone”.\n\n\nPharmacologic therapy\n\nDopamine receptor blocking drugs (neuroleptics) and dopamine-depleting drugs have been traditionally used in the treatment of TS even though functional imaging studies or postmortem studies regarding striatal dopaminergic hyperinnervation in TS have been inconclusive32. Although the only two approved therapies for TS by the U.S. Food and Drug Administration (FDA) are haloperidol and pimozide, many other agents are currently used or are in development for the treatment of TS33,34. While the benefit of haloperidol and pimozide for the treatment of TS has been demonstrated in multiple trials, their use is limited by their short- and long-term side effects, including sedation, weight gain and other metabolic complications, hyperprolactinemia, acute dystonic reactions, parkinsonism, tardive dyskinesia, and neuroleptic malignant syndrome35. Other neuroleptics with potentially fewer side effects have been studied in TS. Fluphenazine, a typical antipsychotic with D1- and D2-receptor blocking activity, has also been shown to be effective for TS in small studies. A recent retrospective review of the long-term efficacy and safety of fluphenazine for the treatment of TS was conducted at Baylor College of Medicine from 1985 to 201136. This study included 268 TS patients treated with fluphenazine for an average of 2.6 ± 3.2 years (range 0.01–16.8, 40 patients over 5 years and 13 patients over 10 years). The mean age of initiation was 15.8 ± 10.8 years (range 4.1–70.2) and the mean dose at last follow-up was 3.2 ± 2.3 mg (range 0.5–12 mg) per day. Response to fluphenazine was rated as “moderate to marked” in 80.5% of patients at initial visit and 76% at last follow-up. The most common side effects were drowsiness (26.1%), weight gain (11.6%), akathisia (8.5%), and acute dystonic reactions (7.0%); there were no cases of tardive dyskinesia.\n\nAtypical antipsychotics such as risperidone and aripiprazole have been studied in open-label and randomized, placebo-controlled studies for the treatment of TS35. A meta-analysis of antipsychotics for the treatment of TS found no difference in efficacy among risperidone, haloperidol, pimozide, and ziprasidone37. Side effects such as sedation, weight gain, and hyperprolactinemia have limited their tolerability35. Aripiprazole, a third-generation antipsychotic with D2-receptor antagonist and agonist properties, has been studied for TS in case series and open label studies. The largest case series of aripiprazole for the treatment of TS included 100 patients treated between 2005 and 201038. These patients (mean age of 27.1 ± 11.5 years) were treated with a mean daily dose of 17.0 ± 9.6 mg. At first follow-up after 1–6 months, 82% had moderate or marked reduction in tic severity and this was sustained in all but one patient after 12-month follow-up. Five patients reported an improvement in comorbid psychiatric disorders including anxiety, depression, aggression, and ADHD, which may be partly due to aripiprazole’s effect on the serotonergic system. Adverse effects were reported in 51% of subjects, with the most common being drowsiness, agitation, sleep disturbance, and nausea. A randomized, double-blind, placebo-controlled trial of aripiprazole included 61 children and adolescents with TS39. After 10 weeks, there was significantly greater improvement in the Yale Global Tic Severity Scale (YGTSS) total tic score compared to baseline in the treatment arm compared to placebo (-15.0 vs. 9.6, respectively, p=0.0196). Side effects of aripiprazole included significant increase in mean body weight, body mass index, and waist circumference. A systematic review of aripiprazole for TS included six randomized controlled trials and found a similar efficacy of aripiprazole and haloperidol for TS but with significantly less risk of drug-induced movement disorders with aripiprazole (1.5%) vs. haloperidol (43.5%)40. The most common adverse effects attributed to aripiprazole were nausea/vomiting (5.5%), drowsiness (3.9%), headache (3.5%), and dizziness (3.5%). Although the drug has been promoted as having a low risk of tardive dyskinesia, this iatrogenic complication has been reported with all typical and atypical neuroleptics (with a possible exception of clozapine), including aripiprazole41.\n\nIn addition to neuroleptics approved by the FDA for the treatment of psychiatric disorders, there are novel, experimental, anti-dopaminergic drugs currently being investigated in the treatment of TS. Ecopipam, a selective D1-receptor antagonist, was evaluated in 18 adult TS patients in an open-label study42. There was significant improvement in the primary endpoint, a change in YGTSS total tic score at 8 weeks (25.3) compared to baseline (30.6) (p=0.0004). The most common adverse events were sedation (39%), fatigue (33%), insomnia, (33%), somnolence (28%), anxiety (22%), headache (22%), and muscle twitching (22%). This drug has the potential to improve tics, but it is not yet clear whether its side effects will include those typically associated with D2-receptor blockade, including tardive dyskinesia. A multi-center, randomized, placebo-controlled study of ecopipam in children with TS is currently underway in several North American centers (ClinicalTrials.gov registration: NCT02102698).\n\nTetrabenazine, a dopamine-depleting drug that acts by inhibiting vesicular monoamine transporter 2 (VMAT2), has been shown to be effective in open-label trials for the treatment of TS43,44. A retrospective study conducted at Baylor College of Medicine evaluated the response to tetrabenazine in patients with various hyperkinetic movement disorders including 92 with tics (mean age 24.1 years)43. Moderate to marked improvement in tics was noted by 76.7% of patients. The most common adverse effects reported, all dose-related, were drowsiness (25.0%), parkinsonism (15.4%), depression (7.6%), and akathisia (7.6%). An open-label prospective study of tetrabenazine for TS included 120 patients45. Patients received a mean daily dose of 70.5 mg for a mean duration of 19 months. There was improvement in the Clinical Global Impression of Change scale in 76% of patients at the final visit and side effects occurred in 2–5% of patients (somnolence, depression, asthenia, parkinsonism, and akathisia). In the United States, tetrabenazine (now available as a generic drug) carries a black-box warning regarding the risk of depression and suicidality. This potential side effect seems to be more common in patients with a pre-existing history of depression46. In contrast to the dopamine receptor blocking drugs, tetrabenazine does not appear to cause tardive dyskinesia and may have fewer other side effects, including weight gain47. Other dopamine depletors currently being investigated in the treatment of TS include SD-809 (deutetrabenazine), a deuterated form of tetrabenazine, which is also being investigated in tardive dyskinesia and Huntington disease (Clinicaltrials.gov registration: NCT01795859), and NBI-98854, purified parent drug of the (+)-α-isomer of tetrabenazine (valbenazine), (Clinicaltrials.gov: NCT02581865). In a pilot, open-label study, 23 adolescent patients (mean age 16 years; range: 12–18) with moderate-to-severe tics associated with TS were titrated over a 6-week period and maintained for 2 weeks at a mean dose of 32.1 mg (range: 18–36 mg) of SD-80934. An independent blinded rater assessed tic severity using the YGTSS and tic impact using the TS-Clinical Global Impression. The mean YGTSS total tic score improved by 37.6% (p<0.0001) and there were also significant improvements in secondary outcome measures. No serious or severe adverse effects were reported, but one subject withdrew from the study for an adverse effect of irritability that was unrelated to the study drug. These VMAT inhibitors, SD-809 and NBI-98854, have a more favorable pharmacokinetic and side effect profile than tetrabenazine, as suggested by some preliminary studies in Huntington disease and tardive dyskinesia, although further studies are needed.\n\nA variety of antiepileptic medications have been studied in TS48. A meta-analysis of topiramate for TS identified 14 trials involving 1003 TS patients49. The included studies had either haloperidol (12 studies) or tiapride (two studies) as the control. There were many limitations to this analysis, including overall poor quality of the randomized trials. A meta-analysis of three trials that used the YGTSS to evaluate tic severity found a significant improvement in this scale favoring topiramate compared to control treatment. In a meta-analysis of nine studies evaluating improvement of tics by >50%, there was no significant difference between the topiramate and control groups. A multi-center, placebo-controlled study of topiramate in TS involving 29 patients (mean age of 16.5 years) showed a significantly greater improvement in the total tic score of the YGTSS at day 70 compared to baseline in the topiramate group (mean dose 118 mg) vs. placebo (-14.29 vs. 5.00, p=0.0259)50. There was also significant improvement in the clinical global impression and premonitory urge without a difference in adverse events between the groups. Levetiracetam, another anticonvulsant, has been shown to be helpful in the treatment of tics in open-label studies, but randomized controlled studies failed to demonstrate its benefit48.\n\nAlpha agonists such as guanfacine and clonidine have been found to be useful in the treatment of mild tics and may have a particular benefit in patients with co-existing ADHD and impulse control disorder37. A meta-analysis of published studies of alpha-2 agonists for the treatment of TS found a medium-to-large effect on tics in subjects who had co-existing ADHD, but only a small non-significant benefit on tics in studies that excluded co-existing ADHD37. Potential side effects of these medications include sedation, light-headedness, headaches, and irritability.\n\nBotulinum toxin may be helpful in the treatment of focal motor tics and in some simple and complex phonic tics (including coprolalia)51. Open-label studies and cases series have demonstrated the benefits of botulinum toxin not only in ameliorating the intensity of the tics but also in reducing the frequency and the regional premonitory urge52. A randomized, double-blind, placebo-controlled study of botulinum toxin for motor tics demonstrated a significant improvement in urge and tic frequency with botulinum toxin53. This small study showed no significant differences in other measures, such as severity score, tic suppression, pain, and patient global impression, possibly because of its small sample size, relatively mild symptoms, and a single treatment protocol which does not reflect the clinical practice of evaluating patients after several adjustments in doses and sites of injections. A report of 30 TS patients treated with botulinum toxin for phonic tics reported an improvement in 93% of patients with 50% being free of phonic tics54. Premonitory sensation and quality of life also improved. The most common side effect was hypophonia in 80% of patients. Botulinum toxin can be helpful in targeting a few particularly bothersome tics that are refractory to oral medications, such as repetitive cervical extension (so called “whiplash tics”) seen in some cases with malignant TS that can be associated with subsequent complications such as compressive cervical myelopathy55.\n\nThere is a growing public interest in cannabinoids for the treatment of movement disorders including TS56. Anecdotally, patients often report an improvement in tics and some behavioral symptoms with cannabis57,58. In two small controlled trials, an improvement in tics was demonstrated with delta-9-tetrahydrocannabinol (THC) without major side effects57. According to a Cochrane review on the efficacy of cannabinoids in TS, definitive conclusions about the safety and efficacy of cannabinoids in the treatment of TS cannot be drawn59. Further studies with a larger number of patients and of longer duration are needed to determine the efficacy and safety of cannabinoids for the treatment of TS56.\n\nNew therapies for TS are being studied in clinical trials including AZD5213, a histamine H3 receptor antagonist. Given the possible association between mutations in the HDC gene and TS, this drug may be promising for TS.\n\n\nBehavioral therapy\n\nBehavioral therapy is another treatment option for TS and its efficacy has been demonstrated in a number of randomized controlled trials. One study compared behavioral therapy to supportive therapy and education (control group) in children aged 9–17 years with TS or chronic tic disorder60. This study included 126 children who were randomized to eight treatments of behavioral therapy over 10 weeks or control group. Three monthly booster sessions were conducted for those who responded. The behavioral therapy consisted of comprehensive behavioral intervention for tics (CBIT), which is based on habit reversal therapy and includes other components such as relaxation techniques. A blinded examiner evaluated the subjects. After 10 weeks of treatment, the behavioral intervention resulted in a significantly greater reduction in the YGTSS (24.7 to 17.1) from baseline to endpoint compared with the control treatment (24.6 to 21.1, p<0.001) with a difference between groups of 4.1. On the Clinical Global Impression–Improvement Scale, the behavioral intervention group was more likely to be rated as “very much improved” or “much improved” compared to the control group (52.5% vs. 18.5%, p<0.001). Only 12/126 subjects did not complete the study. These outcomes were independent of tic severity. Of the initial responders who were evaluated at 6 months, 87% had a persistent response to behavioral therapy. A second randomized controlled trial evaluated the benefit of CBIT, this time in adult patients with TS or chronic tic disorder61. In this study, 122 subjects were randomized to eight sessions of CBIT or eight sessions of supportive therapy over 10 weeks. At 3 months, there was significantly greater mean reduction compared to baseline in the YGTSS in the CBIT group (24.0 to 17.8) compared with the control group (21.8 to 19.3) (p<0.001). In the treatment group, 38.1% was rated as “much improved” or “very much improved” on the Clinical Global Impression–Improvement scale vs. 6.4% in the control group. The dropout rate was 13.9%, and for those available for assessment, there was persistent benefit at 6 months. A 2014 meta-analysis of the randomized controlled trials of behavioral therapy in TS identified eight trials with a total of 438 participants62. There was a medium-to-large effect size for behavioral therapy compared to comparison conditions, and the number needed to treat was three. Increasing age, a greater number of treatment sessions, and less co-occurring ADHD was associated with a greater effect size. While this therapeutic intervention may be helpful for certain patients, as with all treatment options for TS, there are some limitations to this form of therapy, including the amount of effort and compliance by the patient (and parents) needed for this therapy to provide sustained benefit, limited access to providers trained in the technique, and lack of insurance coverage for the treatment63.\n\n\nSurgical therapy\n\nDeep brain stimulation (DBS) can be effective for patients with medication and behaviorally refractory TS with disabling tics. In one retrospective study, which included 13 patients with refractory TS treated with globus pallidus internus (GPi) DBS at follow-up of a mean 41.9 months (range 13–80 months), there was a 52.1% improvement in YGTSS compared to baseline64. Furthermore, there was mean improvement of 45.7% in the Gilles de la Tourette Syndrome-Quality of Life Scale score. In another study involving 48 patients with refractory TS treated with mostly thalamic DBS, in which 37 subjects completed the study, 78% had a reduction of greater than 50% on the YGTSS65. A double-blind, crossover trial enrolled 15 patients with refractory TS to evaluate GPi DBS66. Subjects were randomized to stimulation on or off for the first 3 months and then switched to alternative treatment. The patients and evaluating clinicians were blinded to the treatment group. There was a mean improvement of 12.4 points (p=0.048) on the YGTSS total score in the on-stimulation period compared to off-stimulation period. During the open-label phase, there was a greater improvement in YGTSS total scores, possibly reflecting the influence of patient expectations on outcomes. There were three serious adverse events, two hardware infections, and one DBS-induced hypomania.\n\nThe two most common targets studied for DBS in TS are the thalamus (centromedian parafascicular complex, centromedian nucleus-substantia periventricularis-nucleus ventro-oralis internus) and the postero-ventrolateral and antero-medial regions of the GPi67. However, multiple other targets have been tried including the anterior limb of the internal capsule, nucleus accumbens, subthalamic nucleus, and the globus pallidus externus. Potential side effects of DBS in general include intracerebral hemorrhage, ischemia, and infection. There have been reports of higher rates of infection (18%) in DBS for TS thought to be due to compulsive touching of the surgical scar in TS patients68. Psychiatric symptoms are also possible, including worsening depression, hypomania, psychosis, anxiety, and agitation67. A small study involving five TS patients demonstrated the feasibility and improvement of “scheduled” DBS at regular intervals for tic severity69. This was a proof-of-concept study of scheduled DBS therapy as opposed to the classic continuous stimulation. Future techniques of neuromodulation may focus on closed-loop stimulation that adjusts stimulation parameters based on neural feedback and thus providing more effective and efficient therapy70. A panel of experts reviewed 48 studies of DBS for TS published since 1999 and proposed consensus guidelines71. The recommendations of the panel were as follows: 1. DBS should be considered in TS patients with disabling tics with a YGTSS >35/50; 2. patients should have tried medications from at least three pharmacologic categories − alpha-adrenergic agonists, dopamine antagonists, and one additional category; and 3. behavioral therapy, such as CBIT, should be offered to patients prior to surgery. Patients should be evaluated by a multidisciplinary team, and comorbid conditions should be treated and stable.\n\nThe ideal DBS target in TS is still unknown72. The findings of randomized controlled studies of DBS in TS are difficult to assess because of the fluctuating nature of tics, the lack of standardized programming strategies, and the relatively long duration needed for tic control68,73.\n\nThe optimal treatment strategy for TS patients must take into consideration the severity of tics and their effect on daily functioning and quality of life (Table 1 and Table 2)34,74. Behavioral therapy may be limited by access to trained providers. Various pharmacologic treatments including oral medications and botulinum toxin should be selected based on tic severity and comorbid conditions, keeping side effect profiles of these medications in mind. Finally, DBS should be reserved for those with severe tics that are refractory to conventional therapy.\n\nVMAT2 = vesicular monoamine transporter 2\n\nDBS = deep brain stimulation\n\n\nConclusions\n\nTS is a complex disorder with motor manifestations (motor and phonic tics) and a variety of behavioral co-morbidities (ADHD, OCD, impulse control disorders, and others). While there have been advances in our understanding of the neural networks involved in TS, particularly as a result of new imaging techniques, the mechanisms of the disturbed pathways are still not well understood. Furthermore, despite a strong heritability for the disorder, a causative gene or genes have yet to be identified. Various treatment options are available for TS and need to take into consideration tic severity, effect on quality of life, and comorbid disorders. Further study of DBS for medically refractory malignant TS is needed in order to determine the most effective target and optimal programming strategies. New medical and surgical treatments are currently being studied in order to provide better quality of life in patients with TS.\n\n\nAbbreviations\n\nADHD = attention deficit/hyperactivity disorder\n\nCI = confidence interval\n\nCBIT = comprehensive behavioral intervention for tics\n\nDBS = deep brain stimulation\n\nFDA = Food and Drug Administration\n\nfMRI = functional magnetic resonance imaging\n\nGABA = γ-aminobutyric acid\n\nGPi = globus pallidus internus\n\nGWAS = genome-wide association study\n\nHDC = histamine decarboxylase\n\nOCD = obsessive-compulsive disorder\n\nOR = odds ratio\n\nTS = Tourette syndrome\n\nSMA = supplementary motor area\n\nSNPs = single nucleotide polymorphisms\n\nVMAT2 = vesicular monoamine transporter 2\n\nYGTSS = Yale Global Tic Severity Scale", "appendix": "Competing interests\n\n\n\nDr. Thenganatt has no relevant disclosures. Dr. Jankovic has served as a consultant or as an advisory committee member for Allergan, Inc; Auspex Pharmaceuticals, Inc; Teva Pharmaceutical Industries Ltd.\n\n\nGrant information\n\nDr. Jankovic has received research grants from Allergan, Inc; Auspex Pharmaceuticals, Inc; Lundbeck Inc; Medtronic; Merz Pharma; Psyadon Pharmaceuticals, Inc; St. Jude Medical; Teva Pharmaceutical Industries Ltd.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nJankovic J, Gelineau-Kattner R, Davidson A: Tourette's syndrome in adults. Mov Disord. 2010; 25(13): 2171–2175. PubMed Abstract \| Publisher Full Text\n\nPatel N, Jankovic J, Hallett M: Sensory aspects of movement disorders. Lancet Neurol. 2014; 13(1): 100–112. PubMed Abstract \| Publisher Full Text\n\nBaizabal-Carvallo JF, Jankovic J: Speech and voice disorders in patients with psychogenic movement disorders. 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PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nWorbe Y, Lehericy S, Hartmann A: Neuroimaging of tic genesis: Present status and future perspectives. Mov Disord. 2015; 30(9): 1179–1183. PubMed Abstract \| Publisher Full Text\n\nBlack KJ, Jankovic J, Hershey T, et al.: Progress in research on Tourette syndrome. J Obsessive Compuls Relat Disord. 2014; 3(4): 359–362. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMuellner J, Delmaire C, Valabrégue R, et al.: Altered structure of cortical sulci in gilles de la Tourette syndrome: Further support for abnormal brain development. Mov Disord. 2015; 30(5): 655–661. PubMed Abstract \| Publisher Full Text\n\nMartín-Rodríguez JF, Ruiz-Rodríguez MA, Palomar FJ, et al.: Aberrant cortical associative plasticity associated with severe adult Tourette syndrome. Mov Disord. 2015; 30(3): 431–435. PubMed Abstract \| Publisher Full Text\n\nSuppa A, Marsili L, Di Stasio F, et al.: Cortical and brainstem plasticity in Tourette syndrome and obsessive-compulsive disorder. Mov Disord. 2014; 29(12): 1523–1531. PubMed Abstract \| Publisher Full Text\n\nWorbe Y, Marrakchi-Kacem L, Lecomte S, et al.: Altered structural connectivity of cortico-striato-pallido-thalamic networks in Gilles de la Tourette syndrome. Brain. 2015; 138(Pt 2): 472–482. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nYael D, Vinner E, Bar-Gad I: Pathophysiology of tic disorders. Mov Disord. 2015; 30(9): 1171–1178. PubMed Abstract \| Publisher Full Text\n\nNeuner I, Werner CJ, Arrubla J, et al.: Imaging the where and when of tic generation and resting state networks in adult Tourette patients. Front Hum Neurosci. 2014; 8: 362. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nTinaz S, Belluscio BA, Malone P, et al.: Role of the sensorimotor cortex in Tourette syndrome using multimodal imaging. Hum Brain Mapp. 2014; 35(12): 5834–5846. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nCheng B, Braass H, Ganos C, et al.: Altered intrahemispheric structural connectivity in Gilles de la Tourette syndrome. Neuroimage Clin. 2014; 4: 174–181. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nLerner A, Bagic A, Simmons JM, et al.: Widespread abnormality of the γ-aminobutyric acid-ergic system in Tourette syndrome. Brain. 2012; 135(Pt 6): 1926–1936. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nKataoka Y, Kalanithi PS, Grantz H, et al.: Decreased number of parvalbumin and cholinergic interneurons in the striatum of individuals with Tourette syndrome. J Comp Neurol. 2010; 518(3): 277–291. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nTinaz S, Malone P, Hallett M, et al.: Role of the right dorsal anterior insula in the urge to tic in Tourette syndrome. Mov Disord. 2015; 30(9): 1190–1197. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nHanna PA, Janjua FN, Contant CF, et al.: Bilineal transmission in Tourette syndrome. Neurology. 1999; 53(4): 813–818. PubMed Abstract \| Publisher Full Text\n\nPauls DL, Fernandez TV, Mathews CA, et al.: The Inheritance of Tourette Disorder: A review. J Obsessive Compuls Relat Disord. 2014; 3(4): 380–385. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMataix-Cols D, Isomura K, Pérez-Vigil A, et al.: Familial Risks of Tourette Syndrome and Chronic Tic Disorders. A Population-Based Cohort Study. JAMA psychiatry. 2015; 72(8): 787–793. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nDeng H, Gao K, Jankovic J: The genetics of Tourette syndrome. Nat Rev Neurol. 2012; 8(4): 203–213. PubMed Abstract \| Publisher Full Text\n\nErcan-Sencicek AG, Stillman AA, Ghosh AK, et al.: L-histidine decarboxylase and Tourette's syndrome. N Engl J Med. 2010; 362(20): 1901–1908. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKaragiannidis I, Dehning S, Sandor P, et al.: Support of the histaminergic hypothesis in Tourette syndrome: association of the histamine decarboxylase gene in a large sample of families. J Med Genet. 2013; 50(11): 760–764. PubMed Abstract \| Publisher Full Text\n\nScharf JM, Yu D, Mathews CA, et al.: Genome-wide association study of Tourette's syndrome. Mol Psychiatry. 2013; 18(6): 721–728. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPaschou P, Yu D, Gerber G, et al.: Genetic association signal near NTN4 in Tourette syndrome. Ann Neurol. 2014; 76(2): 310–315. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcGrath LM, Yu D, Marshall C, et al.: Copy number variation in obsessive-compulsive disorder and Tourette syndrome: a cross-disorder study. J Am Acad Child Adolesc Psychiatry. 2014; 53(8): 910–919. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYu D, Mathews CA, Scharf JM, et al.: Cross-disorder genome-wide analyses suggest a complex genetic relationship between Tourette's syndrome and OCD. Am J Psychiatry. 2015; 172(1): 82–93. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlbin RL, Koeppe RA, Wernette K, et al.: Striatal [11C]dihydrotetrabenazine and [11C]methylphenidate binding in Tourette syndrome. Neurology. 2009; 72(16): 1390–1396. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTermine C, Selvini C, Rossi G, et al.: Emerging treatment strategies in Tourette syndrome: what's in the pipeline? Int Rev Neurobiol. 2013; 112: 445–480. PubMed Abstract \| Publisher Full Text\n\nJankovic J: Therapeutic developments for tics and myoclonus. Mov Disord. 2015; 30(11): 1566–1573. PubMed Abstract \| Publisher Full Text\n\nMogwitz S, Buse J, Ehrlich S, et al.: Clinical pharmacology of dopamine-modulating agents in Tourette's syndrome. Int Rev Neurobiol. 2013; 112: 281–349. PubMed Abstract \| Publisher Full Text\n\nWijemanne S, Wu LJ, Jankovic J: Long-term efficacy and safety of fluphenazine in patients with Tourette syndrome. Mov Disord. 2014; 29(1): 126–130. PubMed Abstract \| Publisher Full Text\n\nWeisman H, Qureshi IA, Leckman JF, et al.: Systematic review: pharmacological treatment of tic disorders--efficacy of antipsychotic and alpha-2 adrenergic agonist agents. Neurosci Biobehav Rev. 2013; 37(6): 1162–1171. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nWenzel C, Kleimann A, Bokemeyer S, et al.: Aripiprazole for the treatment of Tourette syndrome: a case series of 100 patients. J Clin Psychopharmacol. 2012; 32(4): 548–550. PubMed Abstract \| Publisher Full Text\n\nYoo HK, Joung YS, Lee JS, et al.: A multicenter, randomized, double-blind, placebo-controlled study of aripiprazole in children and adolescents with Tourette's disorder. J Clin Psychiatry. 2013; 74(8): e772–80. PubMed Abstract \| Publisher Full Text\n\nZheng W, Li XB, Xiang YQ, et al.: Aripiprazole for Tourette's syndrome: a systematic review and meta-analysis. Hum Psychopharmacol. 2016; 31(1): 11–8. PubMed Abstract \| Publisher Full Text\n\nPeña MS, Yaltho TC, Jankovic J: Tardive dyskinesia and other movement disorders secondary to aripiprazole. Mov Disord. 2011; 26(1): 147–152. PubMed Abstract \| Publisher Full Text\n\nGilbert DL, Budman CL, Singer HS, et al.: A D1 receptor antagonist, ecopipam, for treatment of tics in Tourette syndrome. Clin Neuropharmacol. 2014; 37(1): 26–30. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nKenney C, Hunter C, Jankovic J: Long-term tolerability of tetrabenazine in the treatment of hyperkinetic movement disorders. Mov Disord. 2007; 22(2): 193–197. PubMed Abstract \| Publisher Full Text\n\nChen JJ, Ondo WG, Dashtipour K, et al.: Tetrabenazine for the treatment of hyperkinetic movement disorders: a review of the literature. Clin Ther. 2012; 34(7): 1487–1504. PubMed Abstract \| Publisher Full Text\n\nPorta M, Sassi M, Cavallazzi M, et al.: Tourette's syndrome and role of tetrabenazine: review and personal experience. Clin Drug Investig. 2008; 28(7): 443–459. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nKenney C, Hunter C, Mejia N, et al.: Is history of depression a contraindication to treatment with tetrabenazine? Clin Neuropharmacol. 2006; 29(5): 259–264. PubMed Abstract \| Publisher Full Text\n\nJankovic J, Clarence-Smith K: Tetrabenazine for the treatment of chorea and other hyperkinetic movement disorders. Expert Rev Neurother. 2011; 11(11): 1509–1523. PubMed Abstract \| Publisher Full Text\n\nCavanna AE, Nani A: Antiepileptic drugs and Tourette syndrome. Int Rev Neurobiol. 2013; 112: 373–389. PubMed Abstract \| Publisher Full Text\n\nYang CS, Zhang LL, Zeng LN, et al.: Topiramate for Tourette's syndrome in children: a meta-analysis. Pediatr Neurol. 2013; 49(5): 344–350. PubMed Abstract \| Publisher Full Text\n\nJankovic J, Jimenez-Shahed J, Brown LW: A randomised, double-blind, placebo-controlled study of topiramate in the treatment of Tourette syndrome. J Neurol Neurosurg Psychiatry. 2010; 81(1): 70–73. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nPersaud R, Garas G, Silva S, et al.: An evidence-based review of botulinum toxin (Botox) applications in non-cosmetic head and neck conditions. JRSM Short Rep. 2013; 4(2): 10. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nKwak CH, Hanna PA, Jankovic J: Botulinum toxin in the treatment of tics. Arch Neurol. 2000; 57(8): 1190–1193. PubMed Abstract \| Publisher Full Text\n\nMarras C, Andrews D, Sime E, et al.: Botulinum toxin for simple motor tics: a randomized, double-blind, controlled clinical trial. Neurology. 2001; 56(5): 605–610. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nPorta M, Maggioni G, Ottaviani F, et al.: Treatment of phonic tics in patients with Tourette's syndrome using botulinum toxin type A. Neurol Sci. 2004; 24(6): 420–423. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nCheung MY, Shahed J, Jankovic J: Malignant Tourette syndrome. Mov Disord. 2007; 22(12): 1743–1750. PubMed Abstract \| Publisher Full Text\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\nMüller-Vahl KR: Treatment of Tourette syndrome with cannabinoids. Behav Neurol. 2013; 27(1): 119–124. PubMed Abstract \| Publisher Full Text\n\nKluger B, Triolo P, Jones W, et al.: The therapeutic potential of cannabinoids for movement disorders. Mov Disord. 2015; 30(3): 313–327. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCurtis A, Clarke CE, Rickards HE: Cannabinoids for Tourette's Syndrome. Cochrane Database Syst Rev. 2009; (4): CD006565. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nPiacentini J, Woods DW, Scahill L, et al.: Behavior therapy for children with Tourette disorder: a randomized controlled trial. JAMA. 2010; 303(19): 1929–1937. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nWilhelm S, Peterson AL, Piacentini J, et al.: Randomized trial of behavior therapy for adults with Tourette syndrome. Arch Gen Psychiatry. 2012; 69(8): 795–803. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nMcGuire JF, Piacentini J, Brennan EA, et al.: A meta-analysis of behavior therapy for Tourette Syndrome. J Psychiatr Res. 2014; 50: 106–112. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nScahill L, Woods DW, Himle MB, et al.: Current controversies on the role of behavior therapy in Tourette syndrome. Mov Disord. 2013; 28(9): 1179–1183. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang JG, Ge Y, Stead M, et al.: Long-term outcome of globus pallidus internus deep brain stimulation in patients with Tourette syndrome. Mayo Clin Proc. 2014; 89(11): 1506–1514. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nServello D, Zekaj E, Saleh C, et al.: Deep Brain Stimulation in Gilles de la Tourette Syndrome: What Does the Future Hold? A Cohort of 48 Patients. Neurosurgery. 2016; 78(1): 91–100. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nKefalopoulou Z, Zrinzo L, Jahanshahi M, et al.: Bilateral globus pallidus stimulation for severe Tourette's syndrome: a double-blind, randomised crossover trial. Lancet Neurol. 2015; 14(6): 595–605. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nFraint A, Pal G: Deep Brain Stimulation in Tourette's Syndrome. Front Neurol. 2015; 6: 170. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nServello D, Sassi M, Gaeta M, et al.: Tourette syndrome (TS) bears a higher rate of inflammatory complications at the implanted hardware in deep brain stimulation (DBS). Acta Neurochir (Wien). 2011; 153(3): 629–632. PubMed Abstract \| Publisher Full Text\n\nOkun MS, Foote KD, Wu SS, et al.: A trial of scheduled deep brain stimulation for Tourette syndrome: moving away from continuous deep brain stimulation paradigms. JAMA Neurol. 2013; 70(1): 85–94. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nAlmeida L, Martinez-Ramirez D, Rossi PJ, et al.: Chasing tics in the human brain: development of open, scheduled and closed loop responsive approaches to deep brain stimulation for Tourette syndrome. J Clin Neurol. 2015; 11(2): 122–131. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchrock LE, Mink JW, Woods DW, et al.: Tourette syndrome deep brain stimulation: a review and updated recommendations. Mov Disord. 2015; 30(4): 448–471. PubMed Abstract \| Publisher Full Text\n\nViswanathan A, Jimenez-Shahed J, Baizabal Carvallo JF, et al.: Deep brain stimulation for Tourette syndrome: target selection. Stereotact Funct Neurosurg. 2012; 90(4): 213–224. PubMed Abstract \| Publisher Full Text\n\nJimenez-Shahed J: Design challenges for stimulation trials of Tourette's syndrome. Lancet Neurol. 2015; 14(6): 563–565. PubMed Abstract \| Publisher Full Text\n\nGilbert DL, Jankovic J: Pharmacological treatment of Tourette syndrome. J Obsessive Compuls Relat Disord. 2014; 3(4): 407–414. Publisher Full Text" }	[ { "id": "12333", "date": "09 Feb 2016", "name": "Andrea Cavanna", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12334", "date": "09 Feb 2016", "name": "Jonathan W. Mink", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12335", "date": "09 Feb 2016", "name": "Mark Hallett", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-152
https://f1000research.com/articles/5-150/v1	09 Feb 16	{ "type": "Opinion Article", "title": "Open drug discovery for the Zika virus", "authors": [ "Sean Ekins", "Daniel Mietchen", "Megan Coffee", "Thomas P Stratton", "Joel S Freundlich", "Lucio Freitas-Junior", "Eugene Muratov", "Jair Siqueira-Neto", "Antony J Williams", "Carolina Andrade", "Daniel Mietchen", "Megan Coffee", "Thomas P Stratton", "Joel S Freundlich", "Lucio Freitas-Junior", "Eugene Muratov", "Jair Siqueira-Neto", "Antony J Williams", "Carolina Andrade" ], "abstract": "The Zika virus (ZIKV) outbreak in the Americas has caused global concern that we may be on the brink of a healthcare crisis. The lack of research on ZIKV in the over 60 years that we have known about it has left us with little in the way of starting points for drug discovery. Our response can build on previous efforts with virus outbreaks and lean heavily on work done on other flaviviruses such as dengue virus. We provide some suggestions of what might be possible and propose an open drug discovery effort that mobilizes global science efforts and provides leadership, which thus far has been lacking. We also provide a listing of potential resources and molecules that could be prioritized for testing as in vitro assays for ZIKV are developed. We propose also that in order to incentivize drug discovery, a neglected disease priority review voucher should be available to those who successfully develop an FDA approved treatment. Learning from the response to the ZIKV, the approaches to drug discovery used and the success and failures will be critical for future infectious disease outbreaks.", "keywords": [ "Zika", "microcephaly", "drug discovery", "flavivirus", "Aedes", "dengue", "yellow fever", "ebola" ], "content": "Background - Zika virus epitomizes a neglected disease\n\nWe did not have to wait too long for the next virus to make global news1. A fast follower to the Ebola virus (EBOV) epidemic that killed over 11,000 in Africa2–4 during 2014–2015, the Zika virus (ZIKV) has been suggested to have pandemic potential5–7. While EBOV was likely not a new name to most, ZIKV only became part of most people’s vocabulary in the past few weeks in the Western world, indicating a lack of knowledge of the virus. The ZIKV is an arthropod-borne flavivirus of the family Flaviviridae, phylogenetically close to dengue virus and yellow fever, transmitted by Aedes mosquitoes8, that usually causes a mild dengue-like illness with possibly fever, joint pains, rash, and/or swollen lymph nodes9 and has been more recently associated with rare Guillain-Barré syndrome10. It was neglected because there are multitudes of viruses and ZIKV had not seemed to cause severe pathology as it is thought to be the case now. However, the dramatic increase in the number of cases of babies born with microcephaly, especially in Brazil11 and possibly associated with this virus, brought the ZIKV to the immediate attention of the West as it has spread. Cases of microcephaly had never been seen as a risk. It is possible that many children did not make it to adulthood if they were infected with ZIKV. It may have been that common. So few women would be infected during pregnancy. Those who were would be few in number and sporadically, not all at once, so the risk, if present, would have been hard to identify. Brazil without immunity in the population saw large numbers infected immediately as the virus was amplified in the population, resulting in thousands of pregnant women infected at once. Initially the Brazilian Ministry of Health advised reporting diagnosed cases of Zika as dengue, since the symptoms were in most of the cases similar to a mild case of the latter. After the association with microcephaly was announced, they revised the advice and recommended reporting the diseases independently, meaning that the official initial numbers of ZIKV incidence are most likely underestimated. The first baby in the USA born with ZIKV occurred on January 16th, 201612. Although not yet scientifically proven13, the relationship between ZIKV infection during pregnancy and microcephaly is strongly suspected14. Unlike EBOV, the ZIKV has travelled around the globe and has affected many countries9. It has also resulted in the World Health Organisation (WHO) moving much faster than they did against EBOV15, although it is unclear whether they are willing to take a leadership role16,17. Which begs the question who is going to manage the global response to the virus? The WHO Director-General declared on February 1st, 2016 that the cluster of microcephaly cases and other neurological disorders reported in Brazil constitutes a Public Health Emergency of International Concern14 (PHEIC), it has therefore been identified as a problem for the entire world to deal with.\n\nWhat is clear is that the ZIKV has been relatively ignored by researchers for over 60 years, with just 150 articles in PubMed at the time of writing since the original description of the virus was published in 195218, although it was originally isolated in 1947 in Uganda, Africa8. While there are some sources of information on ZIKV such as protein sequences etc. (Table 1), a look in the Protein Data Bank (PDB), ChEMBL and PubChem databases is more despairing at the time of writing, with zero crystal structures for proteins from this virus or any assays that deal with targets or in whole cells. This translates to no molecules that have been screened against ZIKV targets and certainly no approved drugs that have been tested either in vitro or in vivo in relevant animal models which are also absent. Analysis of patents also suggests there are no specific molecules identified as active against ZIKV, although there are several patents on compounds for dengue19. Based on these observations, ZIKV should clearly be labeled as a “neglected disease”.\n\nAccording to NIH RePORTER20, there have been no projects funded to date to specifically address ZIKV. The NIH NIAID has responded by suggesting they would consider submissions for grants that address this virus21. There has been an acceleration in the opportunities for research, especially after the declaration of ZIKV as a PHEIC, but given the inherent need for ethical review and design of trials, this process cannot move as quickly as many wish. For example it is unlikely that any NIH funded projects would start until much later in the year, which would delay potential discoveries. So far, the Bill and Melinda Gates Foundation and the Wellcome Trust have not announced any funding for ZIKV research. If we are to address preventing further spread of this virus, we have to move much faster and be coordinated in our response, maybe implementing a disruptive approach to moving towards a potential cure. It appears the NIH is not actively working on their own in vitro assay for ZIKV. Those extramural scientists already funded to work on similar viruses may be in a position to shift resources or perhaps may have access to alternative funding sources that could be utilized. This does beg the question how we can achieve faster dispersal of funding resources that can be mobilized in public health emergencies like this, whether through the WHO or World Bank or others. Dedicated teams of experts that can tackle such challenges perhaps also need to be convened to see if they can help lead global efforts. These need to be initiated in days and not weeks or months. Clearly, this did not happen with EBOV, and so far, it has not happened with ZIKV, at least in the USA.\n\n\nKickstarting ZIKV drug discovery\n\nWe, and others, have already suggested what steps perhaps could be taken to kick start a drug discovery program in such a circumstance as this22. There is significantly more information on the related flavivirus, dengue virus, with several high throughput screens and computational drug discovery efforts that have resulted in small molecule hits (Table 2). If we want a molecule to reach the clinic quickly for ZIKV, probably the most expedient method may be to repurpose FDA and/or EU approved drugs23 (Table 2). This has been an approach that has led to new in vitro or in vivo active compounds of clinical relevance in a number of cases24,25. The challenge, of course, is how to do this when there is zero prior work either in vitro or in silico. We could certainly leverage the data and models (including computational models) that are available for dengue virus as a starting point, but ideally we need to generate some data for compounds screened against the ZIKV. It is possible that there are already data available sitting behind academic or corporate firewalls, and ideally these need to be released to the world for examination (preferably as open data). We now offer some steps which could be taken immediately:\n\nA first step would be to develop a whole cell or target-based ZIKV in vitro assay that would be amenable to medium to high throughput screening. This would need to be undertaken in BSL 2 facilities, which may limit the number of laboratories that can perform the screening, though expedited data sharing could allow others to help with analyses, informatics, contextualization, quality control and related aspects of the work and thereby accelerate it26. In particular, high content screening can considerably speed the discovery and development of new drugs for Zika chemotherapy. By precluding the need for validated targets, cell-based screening enables the discovery of compounds that can inhibit virus entry and/or replication in human cells, by either deploying fluorescent protein-tagged ZIKV or using antibodies as probes for detection of viral proteins expressed in host cells. This approach has been successfully applied to flaviviruses such as Hepatitis C and dengue27. A simpler assay that could be easily implemented in laboratories with isolated virus not requiring expensive automation or instrumentation would be a viability assay for host cells infected with the virus. Viability markers such as resazurin (Sigma R7017) would be an inexpensive viability marker that could be assessed by colorimetric or fluorimetric readout after being converted to resofurin by the mitochondria of the host cell. The assay design could start with plating host cells such as Vero in a micro-well plate and adding the test compounds followed by addition of ZIKV. Adding the compounds before the virus allows for the detection of invasion inhibitors. Incubation for 4 to 5 days would be enough to eliminate all the cells in the wells untreated or with ineffective compounds. Resazurin solution would be added on the last day of the assay and incubated for at least 1 hour. Effective antiviral compounds would prevent cell death and could be detected by the reduction of resazurin to resofurin by the change in color (from purple to pink) or by fluorescence readout with excitation at 515nm. This assay would not require any genetic manipulation of the virus, and could be implemented with different clinical isolates, being also amenable to mid-high throughput scale screening.\n\nA second step that seems appropriate would be to test drugs and other chemical compounds in the assay developed in step 1. We have summarized all the compounds and chemical libraries suggested for testing against ZIKV in Figure 1. We also sorted them by the priority level for testing. The number of chemicals at each level is given in parenthesis. Here we want to emphasize that we strongly support the idea of drug repurposing in general because it is the quickest way to the introduction of a drug into the market and its use in patients23,28. Due to the absence of any relevant treatment, this is especially important for the rapid discovery of a drug against ZIKV. We also suggest to start from the 48 FDA-approved antivirals (Table 2)29,30. Special priority should be given to the antivirals that were shown to be active against other flaviviruses such as dengue virus (Supplementary material S1), yellow fever, Japanese encephalitis, etc., and to a lesser degree, against other members of the flaviviridae family like Hepatitis C (Table 2). In addition to antivirals, we could also recommend approved non-antiviral drugs that have shown antiviral activity. Moreover, being inspired by the discovery of anti-influenza properties of brinzolamide31 and activity of toremifene against Middle East respiratory syndrome coronavirus infection32, and EBOV33 we also recommend to test, in addition to antiviral compounds, all other marketed drugs. This will increase our chances to find a treatment against ZIKV and will preserve all the benefits of drug repurposing. The main reason preventing us from this approach could be potential low throughput of the developed assay. Another obstacle is the cost of these drugs or corresponding drug libraries, e.g., Prestwick Chemical Library34. However, this could be overcome by in-kind donation of drug samples from big pharmaceutical companies (Pfizer, GSK, etc.) or chemical manufacturers (Prestwick, ChemBridge, Selleck, etc.). Other compounds that are not approved drugs could be also tested (Figure 1). We believe it is still better to start from compounds already approved or undergoing clinical trials (e.g. NIH clinical collection) but not yet approved by FDA and chemicals active against dengue, yellow fever, etc. The latter could be found in HTS assays, ChEMBL, PubChem, etc., and are summarized in Table 3. Perhaps less attractive but still a reasonable step is the use of focused libraries of drug-like compounds and, as a last resort, large diverse chemical libraries containing millions of compounds.\n\nA third approach would be to explore the complete genome of ZIKV35,36 or the recently published genome for ZIKV circulating in the Americas37 to apply a target-based chemogenomics approach38 in order to identify approved drugs that may be active against the ZIKV for testing in vitro.\n\nA fourth approach would be to develop homology models for ZIKV proteins that are similar to those targeted by molecules that are active against the dengue virus39–45 (Table 3). This would enable structure-based approaches such as docking to significantly narrow down the number of compounds for eventual testing. As this is one of the most accessible approaches at the present time, as an example, we have used freely available online software to create a homology model of the ZIKV envelope protein (Supplementary material S2) that can be used to dock compounds and score them in order to prioritize compounds for in vitro testing (Figure 2; Supplementary material S3–Supplementary material S6). Alternatively, we could turn to libraries of commercially available, drug-like small molecules for screening in silico and then in vitro. An expedited path to their optimization toward pre-clinical candidates could rely on publicly available computational machine learning models for critical physiochemical and ADME properties that we and others have made available to the scientific community46,47.\n\nA. Complete protein shown as a ribbon diagram (generated in Discovery Studio). B. Pyronaridine shown docked into the subunit A homology model, small molecule colored by atom, protein colored by atom charge. C. 2D interaction plot for pyronaridine.\n\nA fifth step would be to understand the mechanism and target of any compounds derived from whole cell screening and confirm compounds that have on-target activity when identified by target-based in vitro or in silico approaches. This might be enabled by using similarity of molecules that have been identified to have activity against targets in other species using target prediction software48,49. Potentially promising molecules could also be screened against other viruses (flaviviruses or others) to identify whether they can be used across a whole virus class or have even broader antiviral activity.\n\nA final step would be to test compounds in an animal model of ZIKV infection such as the mouse50 initially. It is unclear whether larger animal models have been developed and tested yet. Once a suitable model has been validated, we stress the importance of assaying a significant number of promising early candidates (if they exist) and also examining opportunities for drug repurposing. Despite the significant advances in the biological sciences over the last 70 years, we must not forget that many of our current anti-infectives arose during the World War II drug discovery effort. At that point in history, small molecules were synthesized and tested in vivo with little delay. Certainly while we cannot ignore requisite animal toxicity studies and guidelines when devoting animals to such studies, we must also not ignore the goal post: in vivo demonstration of efficacy.\n\nThe overall proposed workflow for rapid drug discovery against ZIKV is represented in Figure 3. We suggest to start from screening in vitro assay (preferably medium- or high-throughput) development with subsequent testing of approved antivirals or other drugs. If drug repurposing will not work, other compounds could be prioritized for testing by docking-based virtual screening using developed homology model or by geno- and phenotypic analyses. It is also possible that this could also be done in parallel using compounds derived from docking prioritized for testing in vitro. Subsequent steps are traditional for any drug discovery pipeline and include development of animal models, clinical trial, and in case of success, manufacturing, marketing, and distribution of a drug against ZIKV.\n\nExp = experimental; VS = virtual screen.\n\nDespite years of knowing of the dangers of Ebola, research into low-hanging fruit - drugs that are FDA approved and perhaps already even indicated in the care of Ebola patients - was limited. Promising leads among FDA approved drugs were identified by Peter Madrid et al. using in vitro cell culture assays with Ebola51. In light of this paper, Médecins Sans Frontières (MSF) apparently looked at amodiaquine, as it was listed by the WHO as a potential drug, and was already used (as artesunate-amodiaquine) empirically in about half of Ebola patients to treat any malaria infections in suspected or confirmed patients (including almost all patients in Sierra Leone, where it was on the country's protocol). The other patients generally were treated with another malaria medication, artesunate-lumefantrine, which had not been found to have potential activity against Ebola. Work by members of our group showed that a pharmacophore potentially could describe the inhibition of EBOV by amodiaquine and three other compounds identified from published screens52. However, this promising lead in the form of amodiaquine identified before the outbreak was not followed up in initial drug therapy against Ebola. New, unapproved drugs and other therapies, like blood plasma, were prioritized with rather disappointing results53–56. Later, MSF looking retrospectively showed that during a brief period where stocks of artesunate-lumefantrine were depleted in Liberia and were replaced with artesunate-amodiaquine, mortality dropped by 31%57. Interestingly, dengue also has had potential drugs identified among already approved FDA drugs in vitro, including amodiaquine and quinacrine (Table 2).\n\nWe should perhaps also analyze whether vaccines for other viruses (e.g. dengue) may be useful against Zika, although in the past the yellow fever vaccine was not58. Questions to address include whether those previously exposed to dengue virus have an increased propensity to ZIKV.\n\nIn addition to development of a drug for ZIKV, complementary parallel efforts should be undertaken in order to understand the virus structure, function, and especially its burden on the human health, including the mechanisms of its pathogenesis and neurological abnormalities. The scientific community needs further information to clarify the dangers created by ZIKV: (i) is ZIKV the only or the main reason for the rise in microcephaly cases and Guillain-Barre syndrome?; (ii) is ZIKV dangerous only to pregnant women and what is the probability to have the above mentioned disorders while being infected?; (iii) what is the mutation potential of the virus?; etc. We need to make sure we are addressing the most important aspects of the disease and not tilting at windmills.\n\n\nZIKV: An opportunity for collaboration\n\nThe ZIKV represents an example where national and international readiness needs to be addressed. Clearly, this is also an opportunity to demonstrate the impact of scientific collaboration that could benefit from the mediation of open scientific exchange via open data, open interpretation and step-by-step iterative progress. We propose that open access journals set up Zika repositories and that traditional science publishers could also open up their articles on dengue and related flaviviruses as a means to spur more research and make the literature accessible. It will be important to coordinate these scientific responses to avoid repetition and create these open repositories (e.g. Wikidata – Table 1), but as with many crowdsourcing-based approaches, openness itself will be the primary enabler for exchange and progress. While such initiatives in the past have been limited to malaria and tuberculosis59–62, diseases which kill millions annually, they have not been widely applied to emerging viruses and neglected tropical diseases. There is no time like the present to see whether these efforts can be brought to bear with great immediacy. The health and lives of a large population of newborns and children is at risk. This may also lead to improved approaches for the next global pandemic.\n\n\nHow do we incentivize ZIKV drug discovery\n\nTo get more companies and groups working on Zika drug discovery63, we propose that this neglected disease should qualify for a neglected disease priority review voucher64–67. While Filoviridae are covered, (e.g. Ebola since 2014), the flavivirus Zika is not. It would need US Senate approval to extend to additional viruses. This obviously takes some effort but Ebola was added at the height of the outbreak, so it is not impossible. The value of these vouchers and those for pediatric rare diseases68 is continuing to increase, so they add a meaningful financial incentive that could bring companies into the effort.\n\n\nSummary\n\nThe neglected disease space has many issues, including shortage of funds, which has driven us to look for opportunities in using computational approaches69 that have been applied to drug repurposing. The recent ZIKV outbreak reasserts the importance of preparedness for new viruses. While we have a whole array of impressive molecular biology technologies at our disposal, our ability to quickly identify and test molecules that might possess antiviral activity is severely hampered by lack of appropriate in vitro assays in place for Zika. We are starting from scratch even though we have known of this virus for over 60 years. This will be an important test of our ability to organize and ramp up efforts that should have been triggered by the Ebola outbreak. However, there should be a warning from that experience, as the big pharmaceutical companies played no role, and it was left to biotechs to field their highly experimental approaches. So for all of the merging of pharmaceutical companies in the last decade, we are possibly weaker for it. Clearly, they too are not willing to pursue a costly antiviral approach, unless there is a substantial financial incentive, and the priority review voucher may fit the bill. And for all the US government’s efforts at being prepared with organizations like the Biomedical Advanced Research and Development Authority (BARDA), who have invested billions of dollars in vaccine readiness for influenza and emerging threats, we are clearly not ready yet. We have herein proposed several approaches that could be actionable now with a bare minimum of resources and funding. It may be the case that we already have FDA and EU approved drugs that while showing activity against other viruses may have a role to play in further testing for efficacy against Zika. While we could develop incredibly sophisticated in vitro models for the ZIKV, all that may be needed is a simple readout as to whether a compound has antiviral activity or not. It should certainly not be forgotten that single agent therapies can be overcome quickly by drug resistance, and so from the very beginning, we may want to consider combination therapies like those used in HIV70. We can learn from antiviral drug discovery in the past and try not to repeat the same failures again. The health of a future generation may very well depend on the decisions we make now and our willingness to collaborate to find a cure.", "appendix": "Author contributions\n\n\n\nAll authors contributed to the collaborative writing of this project.\n\n\nCompeting interests\n\n\n\nS.E. works for Collaborations in Chemistry, Collaborations Pharmaceuticals, Inc. and Collaborative Drug Discovery, Inc.\n\nD.M. is a contractor for the National Center for Biotechnology Information.\n\n\nGrant information\n\nEM is currently Special Visiting Professor in Federal University of Goias - Brazil (PVE-CNPq, grant #313727/2014-7).\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nSE gratefully acknowledges discussions with Dr. Priscilla L. Yang, Dr. Warren G. Lewis, Dr. Egon Willighagen, Dr. Rajarshi Guha, Dr. Robert Davey, Dr. Wei Zheng and Dr. Rachelle J. Bienstock while Dr. Christopher D. Southan provided assistance patent searching.\n\n\nSupplementary material\n\nSupplementary material S1. Structures of known dengue virus inhibitors as sdf.\n\nSupplementary material S2. Method and results for Homology model and docking for Zika virus envelope protein.\n\nThe following sequence of the Zika virus envelope protein (E) was taken from the polyprotein109, where the part corresponding to E is from residues 291–592, while the IG-like domain III is from residues 601–693 as shown below\n\n\n\nSwiss-Model110 was used to construct a homology model using the Dengue crystal structure 4gsx as a template (58.69 identity)111–114.\n\nThe complete homology model of subunit A was then used in Discovery Studio version 4.1 (Biovia, San Diego, CA) and the ‘prepare protein’ protocol was used before the ‘Dock ligands’ protocol. Selected molecules were initially docked using a docking sphere of 13 angstroms. The proposed binding site was centered on residues 270–277 and a site sphere created (coordinates 17.07, -21.94, 25.70) with 13 Å diameter. The protocol included 10 hotspots and docking tolerance (0.25). The FAST conformation method was also used along with steepest descent minimization with CHARMm. Further parameters followed the default settings. Out of 3 compounds of interest initially docked, pyronaridine had the highest LibDockScore score of 142. Quinacrine – (a known FDA approved drug that has shown activity against Dengue (IC50 0.55μM – 87) has a docking score of 128. The well known antiviral Ribavirin has a docking score of 101. These predictions suggest that Quinacrine may be targeting this protein and that other antimalarials may also be worth testing.\n\nThe Prestwick Chemical Library34 of 1280 molecules was first filtered to remove salts then this was docked in the protein as described above.\n\nThe top 10 docked molecules from the Prestwick Chemical Library (S3), as identified using the best scored conformation, includes 3 antivirals: ritonavir, indinavir and saquinavir. Selected antimalarials from the Prestwick Chemical Library (S4) suggest that these molecules may be worth further testing in vitro versus Zika virus given their availability (alongside pyronaridine and quinacrine).\n\nSupplementary material S3. The top 10 docked molecules from the Prestwick Chemical Library.\n\nSupplementary material S4. Selected antimalarials docked in the Prestwick Chemical Library.\n\nSupplementary material S5. PDB file for homology model.\n\nSupplementary material S6. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "13198", "date": "19 Apr 2016", "name": "Nicholas Meanwell", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 provides a blueprint for a collaborative approach to identifying inhibitors of not only Zika virus but any emerging viral pathogen. Developing a collection of, for example, nucleoside and nucleoside analogue drugs that could be quickly screened would be a useful starting point but developing screens and a detailed understanding of the virus, analogous to that accomplished when the coronavirus SARS emerged, makes considerable sense.", "responses": [] }, { "id": "13487", "date": "20 Apr 2016", "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\nThe article provides a specific set of recommendations for discovering a new small molecule drug to treat Zika virus infection. Figure 1 in the article summarizes this process, beginning with setting up appropriate assays and screening known anti-viral drugs, especially those already characterized against dengue, another member of the family of viruses to which Zika belongs (Figure 3 is a nice summary of the proposed workflow). The authors also suggest ways to incentivize this effort, and organize it as a collaboration. All these strategies make sense and are well precedented and documented in the article. Overall, I recommend the article for approval based on its well-founded analysis of a strategy for addressing a major public health concern.", "responses": [] }, { "id": "13489", "date": "20 Apr 2016", "name": "Wade Blair", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 highlight the urgency and advocate for a collaborative approach to Zika virus drug discovery and development. Advocacy for emerging diseases is always a worthy pursuit and the open drug discovery model suggested by the authors to tackle Zika virus warrants further discussion. The authors go on to outline possible drug discovery strategies, with an emphasis on repurposing FDA or EU approved drugs as a faster path to approval of Zika virus therapeutics. Although this is an attractive approach in theory and should be pursued, it is more likely that lead molecules would be identified from this effort rather than effective Zika therapeutics ready for approval. In this short article format, it is difficult for the authors to adequately cover the different drug discovery approaches they recommend. For example, identifying targets and mechanisms for inhibitors identified from a whole cell screen can be complicated and the authors do not discuss the key challenges typically encountered with such a screening campaign. In addition, one very important aspect that is missing from the discussion is drug safety, particularly given that pregnant women in Zika endemic areas represent the population with the highest unmet medical need. The authors should address the hurdles associated with developing drugs with the requisite safety profile for this vulnerable population.", "responses": [] } ]	1	https://f1000research.com/articles/5-150
https://f1000research.com/articles/5-147/v1	08 Feb 16	{ "type": "Review", "title": "Advances in Understanding Hair Growth", "authors": [ "Bruno A. Bernard" ], "abstract": "In this short review, I introduce an integrated vision of human hair follicle behavior and describe opposing influences that control hair follicle homeostasis, from morphogenesis to hair cycling. The interdependence and complementary roles of these influences allow us to propose that the hair follicle is a true paradigm of a “Yin Yang” type, that is a cold/slow-hot/fast duality. Moreover, a new promising field is emerging, suggesting that glycans are key elements of hair follicle growth control.", "keywords": [ "Glyco-biology", "hair follicle biology", "alopecia", "hair cycling", "glycan" ], "content": "Introduction\n\nThe hair follicle is a true paradigm of mesenchymal-epithelial interaction. From early morphogenesis to a fully formed organ, the hair follicle life-cycle is controlled by a dialog between mesenchymal and epithelial compartments1. However, this dialog relies on a delicate balance between conflicting and/or opposing influences.\n\nWith respect to hair follicle morphogenesis, the reaction-diffusion model explains how slowly diffusing inducers and rapidly diffusing inhibitors orchestrate, through local activation and at distance inhibition, the hair follicle patterned formation. Indeed, the seminal work of A. Turing2 has been recently confirmed through a formal identification of morphogen activator-inhibitor couples, such as Wnt/DKK13 (Figure 1) and EDAR/BMP4.\n\n(A) Wnt morphogen stimulates its own synthesis as well as that of Dkk1, its inhibitor. Wnt diffuses slowly while Dkk1 diffuses rapidly. (B) As a result, in a periodic way, Wnt concentration is higher than that of DKK1, and a hair placode can develop. (C) The reaction-diffusion process thus explains the patterned distribution of hair follicles at the surface of the scalp.\n\nConsidering its dual mesenchymal and epithelial origin, the hair follicle can be considered a composite organ, with a concentric structure. Dermal and epithelial compartments interact with each other and are characterized by specific differentiation programs. Opposing signaling pathways concur to control the unique behavior of human hair follicle and maintain its unique intrinsic homeostasis. As the activity of diffusible factors, such as growth factors and morphogens, can be modulated by glycans, their possible role in hair growth control must be taken into account.\n\n\nHair follicle behavior\n\nThe hair follicle is the only organ in mammals that sequentially and repeatedly transits from a phase of active fiber production (anagen) to a resting phase (telogen), through rapid phases of tissue regression (catagen) and regeneration (neogen). A recently published comprehensive guide describes most of the morphological and immunohistological markers that characterize the different stages of the human hair follicle cycle and the intense tissue remodeling events which take place5. Of note, hair follicle regeneration relies on the cyclical activation of stem cells6. In the human hair follicle, these stem cells are harbored within two distinct reservoirs7,8, one of them bathing in a hypoxic environment9. Instead of a cyclical behavior with an intrinsic automaton, the human hair follicle exhibits a stochastic behavior, the probability of duration of each phase fitting with a lognormal equation10. A new concept (Figure 2) postulates the existence of a bi-stable equilibrium11 which controls human hair follicle dynamics, including an active steady state (the anagen stage) and a resting steady state (the telogen stage), the transition between these two steady states involving either a degradation phase (the catagen phase) or a neo-morphogenesis phase (the neogen phase). It is now believed that mesenchymal and epithelial oscillators control the stochastic autonomous switching between these two steady states12,13. The transition phases are both controlled by a complex and dynamic network of interacting activators and inhibitors, diffusible morphogens, and growth factors of opposite influences14. Of note, however, extrapolating from results only obtained in rodents must be approached with caution, since major differences exist between human and mouse hair follicles in terms of phase duration, synchronicity, tissue remodeling, stem cell reservoirs, and so on.\n\nAn active steady state (ASS) of fiber production (anagen) and a dormant steady state (DSS) (telogen/kenogen) are interspaced by short-lasting phases of regression (catagen) and neomorphogenesis (neogen).\n\nDuring the active steady state, hair fiber production results from a finely, timely, and spatially tuned choreography of gene expression, which is highly sensitive to stimulatory and inhibitory signals. A number of signaling pathways15, cytokines16,17, neuropeptides18, hormones19–22, prostaglandins23, and growth factors24 are known to modulate the duration of the active steady state of the hair follicle (Figure 3). For example, while insulin-like growth factor (IGF)-1 is required for anagen maintenance25,26, fibroblast growth factor (FGF)-5 appears to be a crucial regulator of hair length in humans27, as a strong inducer of the catagen phase. Moreover, the human hair follicle is endowed with an autonomous androgen metabolism28, a strict dependence on arginine29, polyamines30, and glucose31 for growth, and a specific immunological response32. The hair follicle is also endowed with a full prostaglandin metabolism and a complex network of prostaglandin (PG) receptors33,34. Recent data suggest that a delicate equilibrium between PGE2/PGF2a on the one hand and PGD2 on the other hand controls the duration of the active steady state. PGE2/PGF2a promotes hair growth maintenance, while PGD2 inhibits it and triggers anagen to catagen transition35. Finally, re-evaluating the mechanisms by which agents such as cyclosporine A36 or JAK-STAT inhibitors37 promote human hair growth might help to identify new key genes and pathways involved in the control of hair growth.\n\nSummary of diffusible factors having positive (Yang) or negative (Yin) effects on hair growth and cycling.\n\nBesides the active steady state, new data demonstrate that the resting steady state is not as quiescent as suspected and can be divided into a refractory period and a permissive period. Indeed, during the telogen phase, the follicle is under the influence of factors that would repress the onset of the neogen phase and factors that would trigger it. Specifically, a strong expression of bone morphogenetic protein (BMP) and FGF-18 defines the refractory period, during which the neogen onset is prevented. The progressive increase in the production of BMP antagonist noggin, Wnt/Fzz/b-catenin pathway activators, and transforming growth factor (TGF)-β2 then reaches a critical threshold that shifts the telogen follicle to a competency status, receptive to FGF-7, secreted by the nearby dermal papilla, and, ultimately, triggers the onset of the neogen phase38.\n\n\nGlyco-biology of the human hair follicle\n\nIt is clear from the above that the complex and rhythmic behavior of the human hair follicle is under the control of multiple, intricate pathways with opposing influences. In this respect, the interdependence and complementary roles of these influences allow us to propose that the hair follicle is a true paradigm of a “Yin Yang” type duality and harmony. However, in our opinion, the fine tuning of these influences cannot solely rely on the timely and spatially controlled gene expression, but also on glycans, “the third revolution in evolution”39. Glycans are endowed with such a huge molecular diversity that they can be considered the third language of life, after DNA and proteins.\n\nLinear or branched oligosaccharides can be attached to a protein backbone via O-(serine/threonine) or N-(asparagine) linkages. They form the large class of N-Complex type glycans. Glycosaminoglycans are linear copolymers of 6-O-sulfated disaccharide units which define them as chondroitin, dermatan, keratin, or heparin sulfates. Proteoglycans have one or more glycosaminoglycan side chains attached to a core protein. Glycosaminoglycans, proteoglycans, and glycan moieties of glycoproteins have long been known to play important roles in the maintenance of protein conformation and solubility, protection against proteolytic degradation, mediation of biological activity, intracellular sorting and externalization, and embryonic development and differentiation40–45. The distribution of proteoglycans in the human hair follicle was originally described in the early 1990s, namely for chondroitin sulfate, dermatan sulfate, and heparin sulfate proteoglycans46, for syndecan 1, perlecan and decorin47, and for versican48. Thanks to the availability of new immunological tools, the distribution of proteoglycans in the human hair follicle has been further refined49 (Figure 4), highlighting a complex, dynamic, and regionalized network of proteoglycans. With respect to cell surface complex type N-glycans, the use of specific fluorescently labeled lectins (saccharide-binding proteins) revealed a differential N-glycan composition among the different hair follicle compartments50–52 (Figure 5).\n\nDiagram shows the distribution of versican, perlecan, syndecan 1, aggrecan, biglycan, and heparan sulfate proteoglycans in the different hair follicle compartments. BM, basement membrane; CTS, connective tissue sheath; IRS, inner root sheath; ORS, outer root sheath.\n\nDistribution of N-glycans identified by their reactivity with fluorescently labelled Pisum sativum agglutinin (PSA), wheat germ agglutinin (WGA) and Ulex europeus agglutinin (UEA) in both skin and hair follicles. PSA mainly decorates the dermal compartments of skin and hair follicles, while WGA decorates both dermal and epithelial compartments. UEA only decorates the epidermis stratum granulosum and the hair follicle IRS.\n\nWhat could be the role of these glycans? It has been known for quite a long time that growth factor activation could be regulated by proteoglycans53,54 and that heparan sulfate proteoglycans were involved in fine-tuning mammalian physiology55 and in cell signaling during development56. With respect to key regulators of hair follicle growth and cycling, syndecans modulate Wnt signaling cascades57, the glycosaminoglycan chains of proteoglycans shape Hedgehog gradients and signal transduction58, and O-linked glycosylation controls Notch1 interaction with its cognate Delta-like 4 receptor59. Decorin, a small leucine-rich proteoglycan, directly modulates TGF-β, epidermal growth factor (EGF), IGF-1 and hepatocyte growth factor (HGF) signaling, all known actors of hair follicle cycling60, and appears to act as an anagen inducer61. Altogether, these recent results designate glycans as long time ignored key players in hair growth control. But, on top of that, enzymes can further modulate the biological activity of these glycans. For example, fucosyl transferase is absolutely required for Notch activity, and disruption of fucosyl transferase expression in murine hair follicle lineages results in aberrant telogen morphology, a decrease of bulge stem cell markers, a delay in anagen re-entry, and dysregulation of proliferation and apoptosis during the hair cycle transition62. With respect to proteoglycans, heparanase (an endoglycosidase that cleaves heparin sulfate) was found expressed in the outer root sheath of murine hair follicles and identified as an important regulator of hair growth through its ability to release heparin-bound growth factors63. In the human hair follicle, however, heparanase was found located in the inner root sheath. Its inhibition provoked an immediate transition from anagen to catagen64. In this case, the HPSG/heparanase network appears to be a key controller of internal hair follicle homeostasis.\n\nFinally, extracellular sulfatases appear to be critical regulators of heparin sulfate activities. Sulf1 and Sulf2, by removing glucosamine-6S groups from specific regions of heparan sulfate chain, modulate (a) Wnt interaction with its cognate receptor Frizzled, (b) BMP signaling by releasing BMP antagonist Noggin, and (c) FGF-2 ability to form the functional FGF-2-HS-FGFR ternary complex65,66. Of note, TGF-β1, by inducing Sulf1 expression67, might indirectly modulate Wnt, BMP, and FGF-2 activities, which could explain its inhibitory effect on hair growth. From a clinical point of view, alterations of glycosaminoglycan degradation provoke mucopolysaccharidoses and abnormalities in hair morphology68, which can be reversed by appropriate enzyme replacement therapy69.\n\n\nConclusion\n\nThe hair follicle is clearly endowed with a unique behavior. Its bi-stability and the intense remodeling processes that it provokes rely on the permanent dialog between opposing and complementary influences, impacting all follicle compartments. From this interdependent duality, one can easily understand that an optimal way to describe the complex equilibrium which controls hair follicle homeostasis is the concept of “Yin Yang”. Until recently, the understanding of hair growth mainly relied on deciphering the patterns of gene expression within the different hair follicle compartments throughout the hair cycle70,71. From now on, the fine-tuning of the activities of growth factors and morphogens by the modulating effects of glycans will also have to be taken into consideration.\n\nFrom a prospective point of view, it is likely that a better understanding of hair diseases, and more specifically the role of inflammation and immune response in the development of alopecia areata72 and androgenetic alopecia73, will likely provide further insights into the role of the so-called immune privilege74 in hair growth control. Moreover, with the advent of mature metabolomics technologies75 coupled with in vitro human hair growth technology76, one can predict that this integrative approach will permit us to identify these key metabolic pathways sustaining normal hair growth.", "appendix": "Competing interests\n\n\n\nThe author is an employee of L’Oréal company.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nI thank Ms E. 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PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nGeyfman M, Plikus MV, Treffeisen E, et al.: Resting no more: re-defining telogen, the maintenance stage of the hair growth cycle. Biol Rev Camb Philos Soc. 2015; 90(4): 1179–1196. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLauc G, Krištić J, Zoldoš V: Glycans - the third revolution in evolution. Front Genet. 2014; 5: 145. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBoucaut JC, Bernard B, Aubery M, et al.: Concanavalin A binding to amphibian embryo and effect on morphogenesis. J Embryol Exp Morphol. 1979; 51: 63–72. PubMed Abstract\n\nBernard BA, Yamada KM, Olden K: Carbohydrates selectively protect a specific domain of fibronectin against proteases. J Biol Chem. 1982; 257(14): 8549–8554. PubMed Abstract\n\nCodogno P, Bernard B, Font J, et al.: Changes in protein glycosylation during chick embryo development. Biochim Biophys Acta. 1983; 763(3): 265–275. PubMed Abstract \| Publisher Full Text\n\nBernard BA, Newton SA, Olden K: Effect of size and location of the oligosaccharide chain on protease degradation of bovine pancreatic ribonuclease. J Biol Chem. 1983; 258(20): 12198–12202. PubMed Abstract\n\nOlden K, Bernard BA, Humphries M, et al.: Function of glycoprotein glycans. TIBS. 1985; 10(2): 78–82. Publisher Full Text\n\nWang H, Zhou T, Peng J, et al.: Distinct roles of N-glycosylation at different sites of corin in cell membrane targeting and ectodomain shedding. J Biol Chem. 2015; 290(3): 1654–1663. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nWestgate GE, Messenger AG, Watson LP, et al.: Distribution of proteoglycans during the hair growth cycle in human skin. J Invest Dermatol. 1991; 96(2): 191–195. PubMed Abstract\n\nCouchman JR: Hair follicle proteoglycans. J Invest Dermatol. 1993; 101(1 Suppl): 60S-64. PubMed Abstract \| Publisher Full Text\n\ndu Cros DL, LeBaron RG, Couchman JR: Association of versican with dermal matrices and its potential role in hair follicle development and cycling. J Invest Dermatol. 1995; 105(3): 426–431. PubMed Abstract \| Publisher Full Text\n\nMalgouries S, Thibaut S, Bernard BA: Proteoglycan expression patterns in human hair follicle. Br J Dermatol. 2008; 158(2): 234–242. PubMed Abstract \| Publisher Full Text\n\nOhno J, Fukuyama K, Epstein WL: Glycoconjugate expression of cells of human anagen hair follicles during keratinization. Anat Rec. 1990; 228(1): 1–6. PubMed Abstract \| Publisher Full Text\n\nTezuka M, Ito M, Ito K, et al.: Differential analysis of the human anagen hair apparatus using lectin binding histochemistry. Arch Dermatol Res. 1991; 283(3): 180–185. 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PubMed Abstract \| Publisher Full Text\n\nLin X: Functions of heparan sulfate proteoglycans in cell signaling during development. Development. 2004; 131(24): 6009–6021. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nPataki CA, Couchman JR, Brábek J: Wnt Signaling Cascades and the Roles of Syndecan Proteoglycans. J Histochem Cytochem. 2015; 63(7): 465–480. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nWhalen DM, Malinauskas T, Gilbert RJ, et al.: Structural insights into proteoglycan-shaped Hedgehog signaling. Proc Natl Acad Sci U S A. 2013; 110(41): 16420–16425. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nLuca VC, Jude KM, Pierce NW, et al.: Structural biology. Structural basis for Notch1 engagement of Delta-like 4. Science. 2015; 347(6224): 847–853. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nInui S, Itami S: A newly discovered linkage between proteoglycans and hair biology: decorin acts as an anagen inducer. Exp Dermatol. 2014; 23(8): 547–548. PubMed Abstract \| Publisher Full Text\n\nJing J, Wu XJ, Li YL, et al.: Expression of decorin throughout the murine hair follicle cycle: hair cycle dependence and anagen phase prolongation. Exp Dermatol. 2014; 23(7): 486–491. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nLin HY, Kao CH, Lin KM, et al.: Notch signaling regulates late-stage epidermal differentiation and maintains postnatal hair cycle homeostasis. PLoS One. 2011; 6(1): e15842. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nZcharia E, Philp D, Edovitsky E, et al.: Heparanase regulates murine hair growth. Am J Pathol. 2005; 166(4): 999–1008. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nMalgouries S, Donovan M, Thibaut S, et al.: Heparanase 1: a key participant of inner root sheath differentiation program and hair follicle homeostasis. Exp Dermatol. 2008; 17(12): 1017–1023. PubMed Abstract \| Publisher Full Text\n\nLamanna WC, Kalus I, Padva M, et al.: The heparanome--the enigma of encoding and decoding heparan sulfate sulfation. J Biotechnol. 2007; 129(2): 290–307. PubMed Abstract \| Publisher Full Text\n\nSeffouh A, Milz F, Przybylski C, et al.: HSulf sulfatases catalyze processive and oriented 6-O-desulfation of heparan sulfate that differentially regulates fibroblast growth factor activity. FASEB J. 2013; 27(6): 2431–2439. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nYue X, Li X, Nguyen HT, et al.: Transforming growth factor-beta1 induces heparan sulfate 6-O-endosulfatase 1 expression in vitro and in vivo. J Biol Chem. 2008; 283(29): 20397–20407. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMalinowska M, Jakóbkiewicz-Banecka J, Kloska A, et al.: Abnormalities in the hair morphology of patients with some but not all types of mucopolysaccharidoses. Eur J Pediatr. 2008; 167(2): 203–209. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nKloska A, Bohdanowicz J, Konopa G, et al.: Changes in hair morphology of mucopolysaccharidosis I patients treated with recombinant human alpha-L-iduronidase (laronidase, Aldurazyme). Am J Med Genet A. 2005; 139(3): 199–203. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nOhyama M, Kobayashi T, Sasaki T, et al.: Restoration of the intrinsic properties of human dermal papilla in vitro. J Cell Sci. 2012; 125(Pt 17): 4114–4125. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nSennett R, Wang Z, Rezza A, et al.: An Integrated Transcriptome Atlas of Embryonic Hair Follicle Progenitors, Their Niche, and the Developing Skin. Dev Cell. 2015; 34(5): 577–591. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nKang H, Wu WY, Lo BK, et al.: Hair follicles from alopecia areata patients exhibit alterations in immune privilege-associated gene expression in advance of hair loss. J Invest Dermatol. 2010; 130(11): 2677–2680. PubMed Abstract \| Publisher Full Text\n\nMahé YF, Michelet JF, Billoni N, et al.: Androgenetic alopecia and microinflammation. Int J Dermatol. 2000; 39(8): 576–584. PubMed Abstract \| Publisher Full Text\n\nChristoph T, Müller-Röver S, Audring H, et al.: The human hair follicle immune system: cellular composition and immune privilege. Br J Dermatol. 2000; 142(5): 862–873. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "12318", "date": "08 Feb 2016", "name": "Gill Westgate", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12319", "date": "08 Feb 2016", "name": "Rodney Sinclair", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-147
https://f1000research.com/articles/5-146/v1	08 Feb 16	{ "type": "Research Note", "title": "Can ecosystem-scale translocations mitigate the impact of climate change on terrestrial biodiversity? Promises, pitfalls, and possibilities", "authors": [ "Stéphane Boyer", "Bradley S. Case", "Marie-Caroline Lefort", "Benjamin R. Waterhouse", "Stephen D. Wratten", "Bradley S. Case", "Marie-Caroline Lefort", "Benjamin R. Waterhouse", "Stephen D. Wratten" ], "abstract": "Because ecological interactions are the first components of the ecosystem to be impacted by climate change, future forms of threatened-species and ecosystem management should aim at conserving complete, functioning communities rather than single charismatic species. A possible way forward is the deployment of ecosystem-scale translocation (EST), where above- and below-ground elements of a functioning terrestrial ecosystem (including vegetation and topsoil) are carefully collected and moved together. Small-scale attempts at such practice have been made for the purpose of ecological restoration. By moving larger subsets of functioning ecosystems from climatically unstable regions to more stable ones, EST could provide a practical means to conserve mature and complex ecosystems threatened by climate change. However, there are a number of challenges associated with EST in the context of climate change mitigation, in particular the choice of donor and receptor sites. With the aim of fostering discussion and debate about the EST concept, we 1) outline the possible promises and pitfalls of EST in mitigating the impact of climate change on terrestrial biodiversity and 2) use a GIS-based approach to illustrate how potential source and receptor sites, where EST could be trialed and evaluated globally, could be identified.", "keywords": [ "Climate change", "Translocation", "Conservation Biology", "Ecological Restoration", "Terrestrial Ecosystems", "Species Interactions" ], "content": "Introduction\n\nPopulations of animals and plants facing insurmountable barriers to dispersal, and species with low dispersal abilities are likely to be highly impacted by climate change1. With temperatures changing in their historical distribution area, such species may not be able to colonise new habitats with more hospitable climatic conditions and may require human intervention in the form of assisted colonisation if they are to survive2,3.\n\nThe translocation of endangered species has been used as a conservation management tool for several decades4,5 and assisted colonisation as defined by Ricciardi & Simberloff (2009) appears as a logical tool for mitigating the impact of climate change on terrestrial organisms7. However, conservation translocations can be logistically difficult and costly8 and can often fail7,9,10. One potential cause of failure is that the complex community interactions of which the translocated species are a part are left behind. Such interactions are likely to be required for long-term survival of the target species but may not be present or fully functioning at the appropriate rate at the translocation site11. These interactions are also likely to be the first component of the ecosystem to be impacted by climate change12, long before any population or species goes extinct. Conservation strategies should therefore have a greater focus on the translocation of whole ecosystems and their inherent interactions rather than that of individual ‘flagship’ species13. This paper explores a novel approach – ecosystem-scale translocation (EST) – as a means to preserve functioning terrestrial ecosystems threatened by climate change. This involves the careful collection and immediate transfer of topsoil, vegetation and associated ecological communities to a receptor site. Ecosystem-scale translocation may represent an immediately available and practical method for preserving communities and ecosystems threatened by climate change. Here, with the aim of fostering discussion and debate about the EST concept, we 1) outline the possible promises and pitfalls of EST in mitigating the impact of climate change on terrestrial biodiversity and 2) use a GIS-based approach to illustrate how potential source and receptor sites, where EST could be trialed and evaluated globally, could be identified.\n\nSpecies interactions are essential to the functioning of ecosystems. However, in the context of climate change and biodiversity crises, understanding the full range of community interactions that are needed to recreate a functioning ecosystem in a potential receptor area is not realistic14. It follows that ecological engineering through manual planting or regeneration through the seedbank, as is applied in classical restoration programmes15, is not a practical solution for conserving functioning above- and below-ground communities of ecosystems threatened by climate change. On the contrary, the translocation of topsoil, vegetation and all the communities they contain could represent a much-needed shortcut where all components and interactions of an ecosystem are potentially preserved.\n\nUnder the terms habitat translocation, community translocation, vegetation translocation or transplanting, ecosystem translocation has been applied at a small scale to conserve particular plant species or communities impacted by proposed land development16 but also to test the robustness of plant communities in climatically challenging conditions11,17. A number of earlier translocation attempts reviewed by Good et al.18 showed that with appropriate preparation of the receiver sites, translocation of turves produced satisfactory results in terms of plant survival and conservation of community composition for a variety of plant community types. In the mining industry, a similar process called vegetation direct transfer (VDT) has been applied for 30 years as part of mine restoration programmes19–21 sometimes translocating large areas. For example, a total of 75 ha of native grassland, shrubland and low canopy forest have been translocated within the Stockton Mine (New Zealand) in the past 30 years22–24. The method used consists of (i) cutting pieces of land comprising soil, roots and vegetation over a 1–3 m2 area and a depth of 30 cm or more using a digger, (ii) transporting these sods by truck to a new area and (iii) reconstituting the ‘jigsaw’ with minimum gaps between the sods to recreate a continuous habitat (Figure 1). Many studies have highlighted the positive outcomes of such translocations, including: the conservation of plant and beetle communities, soil functions and microbial activity after wetland translocation25,26; the maintenance of soil structure and fertility in translocated grassland; rtebrates (e.g. carabid beetles, weta) and plant species (beech) from shrubland and tussock wetlands22; the preservation of biodiversity and biomass in soil fauna27 and microbial communities28 from alpine vegetation; and the conservation of habitat for birds and invertebrates from translocated forest areas21. There is also an extensive body of work, including peer-reviewed articles21,24,26,29 and reviews18,19,21, but also internal reports and conference presentations (see Supplementary material), most of which point to the conclusion that VDT promotes faster ecosystem recovery than do other conventional restoration techniques such as replanting or hydroseeding.\n\nClockwise: pieces of land comprising vegetation, roots and soil are cut; soil and vegetation sods are loaded on a truck and transported to the receptor site; the landscape jigsaw is then reconstituted with minimum gaps between the sods to recreate a continuous habitat. Credits: Stéphane Boyer and Solid Energy New Zealand Ltd.\n\nCould ecosystem-scale translocation represent a practical and economic translocation method, by which all or most immobile, slow moving and low-dispersal elements of a terrestrial ecosystem are moved together as a functioning community of above- and below-ground organisms? While no taxonomic group is particularly targeted by EST, it is likely that plant, microbial and fungal communities as well as most invertebrates and small vertebrates will directly benefit, while large and mobile terrestrial vertebrates will probably vacate the site due to disturbance during the translocation process. Therefore, EST constitutes a complementary approach to single-species conservation translocation programs, which suffer from a significant taxonomic bias towards larger animals. Indeed, almost 60% of all animal translocation efforts to date have been for large and charismatic species of birds and mammals7, despite these taxa comprising only c. 1% of all known animal species30. Although translocation distances and the sizes of the areas translocated are usually minimised for economic and practical reasons in a restoration context (Figure 2), this process could be scaled up for the purpose of ESTs where the aim is to mitigate the impact of climate change on biodiversity in highly-valued ecosystems. The aim of EST should be to preserve a functioning representative subset of an ecosystem of sufficient size to ensure its long-term survival and functioning. Capturing most ecosystem functions is likely to require the translocation, in stages, of fairly large areas. However, the majority of the current protected areas globally are less than 1,000 ha and places such as Barro Colorado in Panama (1,500 ha), the Bosavi Crater in Papua New Guinea (1,250 ha), or Darwin Island in the Galapagos (110 ha), are good examples of relatively small areas with significant biodiversity interest.\n\nThe orange area corresponds to typical past and current habitat translocation projects, with dots corresponding to published studies (see Supplementary material S1 for a complete list). The green area represents an ideal combination of size and distance for ecosystem-scale translocation, with darker green more suitable, although more difficult to put in place.\n\nOnly a handful of translocation studies have been reported in the scientific literature (Supplementary material) and the extent to which biodiversity conservation was measured in these studies is very limited. More targeted research is therefore required to assess to what extent EST can conserve biodiversity at a range of levels including species, genetic and functional diversity, as well as species interactions and ecosystem functions. Critical factors and recommendations required for habitat translocation include similarity between the environmental conditions of the donor and receptor sites, the translocation technique, and appropriate management of the translocated habitat31. In particular, the nature of the bedrock strata as well as the geochemistry, hydrology and precipitations at the receptor site will have a strong influence on the translocated topsoil. Geology can be measured and matched between donor and receptor sites, while the range of possible changes in fine-scale hydro-dynamics may be much more difficult to predict as climate changes32. One additional and outstanding issue is the selection of a candidate receptor site where the ‘graft’ will not replace communities which are themselves of high-value, possibly leading to the extinction of local species14 or negatively impacting the functioning of surrounding ecosystems6,9. For example, some phytophagous insects could rapidly become invasive when encountering new plant species within their distribution range33. Conversely, the spillover of existing invasive species from areas surrounding the receptor site could negatively impact the graft itself. This is possibly of less concern because species invasion is less likely to happen when translocating complete and functioning ecosystems where most or all niches are occupied and, consequently, where the lack of empty niches make ecosystems more resistant to the establishment of invasive species34. Supposedly, invasion risk could also be reduced if the receptor site is located, within areas where species composition does not differ dramatically from that of the donor site. Nevertheless, it is important to note that any negative impacts are likely to be difficult and costly to reverse after translocation and a thorough risk assessment would be required to ensure that translocated species do not affect the surrounding areas after translocation.\n\nThe cost involved in the translocation of ecosystems is likely to constitute an important limitation. Because only few examples exist, it is difficult to provide a definite price per unit area. One comparative study by Simcock et al.22 showed that translocation in a mine rehabilitation context was seven times more costly than transporting bulldozed soil and vegetation, but twice cheaper than rehabilitation through nursery cultivation followed by manual replanting. This study was based on the translocation of a 0.6 ha of native forest over a few hundred meters distance and most of the additional cost was from transportation between donor and receptor area as trucks could only carry a single layer of sods (i.e. 30 to 50% of full capacity)22. Translocation of larger areas over much longer distances, as required for EST (Figure 2), is likely to be much more costly.\n\nIn addition to a deep understanding of the risks and potential side effects, the application of EST also requires carefully selected receptor sites. Suitable receptor sites should have very low intrinsic biodiversity value because they will be replaced by EST and whatever communities were present will disappear. Such potential sites could be selected in agricultural land that has been marginalised or abandoned due to the erosion of the fertile soil layer and the loss of essential soil nutrients following overexploitation or misuse35. It has recently been estimated that 25% of all terrestrial habitat is highly degraded36, with one hectare of productive land lost globally every 7.67 seconds (www.irri.org). There is growing interest in using such marginal and degraded land to cultivate biofuel crops37,38. However, this land could alternatively be used for biological conservation. When soil is lost or degraded to the extent where growing crops is not possible, the land could still be suitable for receiving EST, because the latter includes topsoil and its associated ecosystem functions. With human-degraded soils scattered across all continents37 potential receptor sites could be available in a range of climate zones.\n\nAssisted colonisation of single species includes moving organisms to habitats with more favorable future climatic conditions5. The timing of assisted colonisation is therefore crucial because those performed before the recipient region is predicted to be climatically suitable are likely to fail39. Determining the “window of opportunity” – a scenario where an ecosystem is still viable at its original location, and where there is an existing suitable receptor site - is likely to prove difficult and will require active adaptive management40. To alleviate the above limitation, EST could be applied to move ecosystems from climatically unstable areas to areas where the climate is projected to remain stable despite global changes41. The climate stability index proposed by Iwamura et al.42 can be used in this context. This index compares observed temperature, precipitation, cloud cover and vapour pressure in 1997–2002 to projected values in 2047–2052. Human-degraded soils that occur in climatically stable areas could offer prime locations to receive ESTs and could receive them from regions of high conservation value (e.g. biodiversity hotspots) currently located in unstable climatic areas.\n\n\nResults\n\nUsing a highly conservative GIS approach (see Method section), we identified a range of potential source and receptor areas at a global scale. The former were located mainly in North America and North Eurasia as well as over the Amazonian forest (Figure 3A), while a large majority of potential receptor areas were located in Africa, with smaller areas also identified in America, the Middle East, Asia and Australasia (Figure 3B). The suitability of these source and receptor areas is largely dependent on the accuracy of current climatic predictive models, which are constantly improving43. Also, climatic stability thresholds and criteria used for selecting highly valuable ecosystems or degraded land can vary greatly. In a similar way to conservation programmes, which often struggle to cross borders44 due to different policy in neighbouring countries and a lack of global legislation, EST is highly unlikely to be applied in a cross-border situation (where an ecosystem with high conservation value would be translocated from one country to another). Therefore, we identified countries where large areas of both source and receptor sites were present (Figure 3C). They include Madagascar, USA (California), Mexico, Turkey, Nigeria, Cameroon, South Africa, Vietnam, Thailand, Myanmar and Indonesia (Table 1). These countries may therefore be ideal candidates to trial EST.\n\nIdeal source areas are those where high value (blue lines) overlaps with low climatic stability (in pink).\n\nIdeal sites are those where high soil degradation (red lines) overlaps with high climatic stability (in purple).\n\nCountries were ranked from best to worst candidates based on size of source area, size of receptor area and minimum distance between the two. Orange background: best candidate countries based on thresholds of 15,000 km2 for the areas and 500 km distance between areas. Numbers in bold correspond to limiting factors. Blue background: countries where distance between source and receptor may be limiting using the above criteria; Purple background: countries where receptor areas may be limiting; Green background: countries where receptor areas may be limiting; Grey background: countries where multiple limiting factors exist. Min. source-receptor distance is the minimum distance between a patch of source area and a patch of receptor area. When source and receptor are adjacent, the minimum source-receptor distance is 0.\n\n\nDiscussion\n\nClear evidence that ecosystems have started to shift in response to climate change is now observable, with plant45 and animal46 advancing towards higher altitudes and latitudes. Using the EST approach, it may be possible to move ecosystems as a preemptive measure to ensure that all immobile, slow moving and low-dispersal elements of a terrestrial ecosystem are moved together as a functioning community of above- and below-ground organisms.\n\nTranslocating communities and ecosystems should not be regarded as a substitute for less invasive conservation efforts when such options are available19,47, but considered only as a last resort, when ecosystems are at inevitable risk of disappearance or collapse. In the review by Bullock (1998), community translocation often did not achieve the preservation of complete and unchanged community, but led to communities ‘which resembles the pre-translocated state in mitigation for the loss of the original community and which retains many of the species found at the donor site. This method should therefore be recommended only when the aim is to mitigate inevitable negative impact on high-valued ecosystems. Important considerations associated with the proposed approach include the differences in bedrock strata, geochemistry and hydro-dynamics between the source and receptor site, the size of the translocated ecosystem subset in relation to edge effects and isolation, and potential negative impacts on the receptor site. These limitations will require careful planning on a case-by-case basis to minimise the risks of failure. However, in the face of the urgency of climate change, there is a pressing need for proposing and testing more proactive and ambitious forms of conservation management. Hoping that most species threatened by climate change will be able to (i) use environmental buffering48; (ii) retreat to refugia49; (iii) follow their climatic niche, potentially with the help of ‘corridors’50; or (iv) exhibit some form of phenotypic plasticity or micro-evolution on very short time-scales51 is unlikely to be sufficient to conserve biodiversity at all scales. Ecosystem-scale translocation adds to the current debate on novel and audacious ideas to stem biodiversity loss, which include de-extinction52 and the setting aside of half the planet for wildlife as proposed by E.O Wilson53. Our analysis provides a starting point for discussion about the potential of EST for the conservation of ecosystems threatened by climate change. Although this is undoubtedly a radical option, and one that should be examined with caution, it could be applied to conserve very highly-valued ecosystems. Even if a number of species or functions were to be lost during the translocation process, this approach would still improve on the current limited single-species conservation translocation strategies. The EST approach is particularly suited to the conservation of small life forms, thereby balancing the current bias toward translocation and conservation of birds and mammals7 and addressing the urgent issue of conserving invaluable invertebrate communities54. This approach has the potential to add a new dimension to the spectrum of conservation tools currently available, with the aim of conserving mature and complex terrestrial ecosystems threatened by climate change.\n\nResearch is urgently required to assess to what extent EST can conserve not only species diversity, but also genetic diversity, functional diversity, species interactions and ecosystem functions. Source and receptor sites that ranked highly in this study would provide ideal candidates to test these hypotheses in medium to large-scale experiments of EST. This information will be critical to inform future EST projects, which are likely to become necessary if we are to tackle one of the biggest ecological challenges of the coming decades: preserving biodiversity at the ecosystem scale in an era of unprecedented climate change.\n\n\nMethod\n\nLiterature included in Figure 2 was selected using the keywords ‘vegetation direct transfer’, ‘habitat translocation’, ‘vegetation translocation’, ‘community translocation’ and ‘vegetation transplant’ in all databases available in ISI Web of Science. This search was restricted to English written articles published from 1910 to 2016 (search conducted in January 2016). The reference lists of papers retrieved using these searches were then screened based on their titles for potential additional references. We used only those articles where the size of area translocated was reported and translocation distance was either given or could be estimated based on maps or GPS coordinates. Additional publications were extracted from the database of Solid Energy New Zealand Ltd. (www.solidenergy.co.nz) and Landcare Research (http://www.landcareresearch.co.nz) using the same search terms. All information used to build Figure 2 is summarised with the corresponding references in the Supplementary material.\n\nWe mapped the location of potential areas where EST may be applicable on a global scale by overlaying the GIS maps described below using ArcGIS 10.1. Potential source areas for EST were identified in GIS as those predicted to have a relatively unstable future climate based on global climate models, while also having high global conservation value. Climatically-unstable areas were identified using an ecoregion-based climate stability index dataset41,55, which incorporates seven different future climatic scenarios, and indicates how different future climate will be compared to present climate. This index ranges from 0.0 to 1.0 where 1.0 corresponds to complete overlap between current and future climates while 0.0 indicates no overlap55. We averaged the climate stability indices across these seven climate scenarios; relatively climatically-unstable ecoregions were identified as those that had a mean stability index of <0.33 (Figure 3A – pink areas). Areas of high global conservation values were identified using the Conservation International Biodiversity Hotspots dataset56 (Figure 3A – blue outlined areas). Potential receptor sites for EST were delineated in the GIS as areas that have both relatively high predicted future climate stability and a high incidence of soil degradation. Climatically stable areas were identified as ecoregions with a mean stability index of >0.66 (Figure 3B – purple areas). Highly-degraded areas (Figure 3B – red outlined areas) were extracted from a global soil degradation GIS dataset (GLASOD57) as those polygons comprising degradation categories 3 and 4. The final composite map (Figure 3C) shows the position of potential source and receptor sites together overlaid with country boundaries. For each country containing both source and receptor sites, we then calculated the total area of, and minimum distances between source and receptor sites within each country, allowing us to rank their potential as candidates for EST (Table 1). We considered countries with source and receptor areas greater than 15,000 km2 as good candidates because they offer a greater range in the choice of possible source and receptor sites. The other characteristic taken into account was the minimum distance between source and receptor areas. When this distance was less than 500 km, countries were ranked highly because road transportation is more likely to be completed within one day, therefore minimising the impact on the integrity of the transported vegetation and soil.", "appendix": "Author contributions\n\n\n\nS.B. developed the concept and designed the manuscript. B.S.C. ran the GIS analysis and produced the maps. S.B. and B.S.C. prepared the figures and tables. S.B., B.S.C, M.-C.L., B.R.W. and S.D.W wrote the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgment\n\nWe are grateful to Solid Energy New Zealand Ltd. and Bathurst Resources Limited for providing information about their restoration programmes as well as internal reports.\n\n\nSupplementary material\n\nData used to construct Figure 2 and additional references.\n\n\nReferences\n\nThomas CD, Cameron A, Green RE, et al.: Extinction risk from climate change. Nature. 2004; 427(6970): 145–8. PubMed Abstract \| Publisher Full Text\n\nFordham DA, Watts MJ, Delean S, et al.: Managed relocation as an adaptation strategy for mitigating climate change threats to the persistence of an endangered lizard. Glob Chang Biol. 2012; 18(9): 2743–55. 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Reference Source" }	[ { "id": "12433", "date": "15 Feb 2016", "name": "Michael J Samways", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 exciting paper which is highly contemporary and challenging to biodiversity conservation in the future. I personally have huge ethical issues with this EST approach as it completely violates sense of place, and also the actual physical aspects are highly embedded in chaos theory, with the chances of predicting outcomes very slim, including the great risks to indigenous biota. Furthermore, even the baselines are changing: with receptor sites under the same stresses as the donor sites, despite the (highly speculative) maps provided here in the MS. However, as futurists we must keep all options open, as the next few decades are going to be very uncertain, and we may have to consider all sorts of strategies which may seem outrageous to us now. While I don’t agree with this EST approach, the authors present some very interesting “promises, pitfalls and possibilities” which although highly contentious are well articulated…and this is the strength of the MS. It is a wonderful trigger for thought and discussion. In short, I recommend the MS be indexed.", "responses": [] }, { "id": "12537", "date": "07 Mar 2016", "name": "John Box", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 exciting but very challenging topic which certainly needs to be raised in order to stimulate the further experimental field trials that are required. Critical success/failure parameters need to be established across the wide range of habitats and communities that could be involved.", "responses": [] } ]	1	https://f1000research.com/articles/5-146
https://f1000research.com/articles/4-618/v1	24 Aug 15	{ "type": "Research Article", "title": "Competition over guarding in the Arabian babbler (Turdoides squamiceps), a cooperative breeder", "authors": [ "Arnon Dattner", "Amotz Zahavi", "Avishag Zahavi", "Arnon Dattner", "Amotz Zahavi" ], "abstract": "Observations on 12 groups comprised of two adult males and one adult female (some included one or two fledglings), tame, individually marked, Arabian babblers (Turdoides squamiceps) in the rift valley in Israel revealed that the babblers compete to guard. The pattern of guarding and the way by which one sentinel replaces another reflect the dominance relationships within the group. The dominant (alpha) male guarded more than any other individual. It interfered with and replaced the guarding by the adult beta male more than it did with the yearlings. About one-third of the replacements occurred less than one minute after the sentinel had assumed guarding. Whereas the dominant often replaced its subordinates directly; subordinates hardly ever replaced their dominants directly. The alpha male often allofed the beta male during the replacement. Replacements and allofeeding of the beta males by the alpha males increased significantly during courtship, when competition over breeding was maximal, and dropped back to their previous level at the start of incubation, highlighting the competitive basis underlying the act of guarding. Competition over altruistic acts, as shown here for guarding, is not compatible with explanations based on the assumption that altruistic acts reduce the fitness (reproductive success) of the altruist. We suggest, in contrast, that by investing in guarding and by intervening in the guarding of its competitors, a babbler demonstrates and signals its quality and its control over its competitors, thereby increasing its prestige and consequently its direct fitness.", "keywords": [ "altruism", "allofeeding", "dominance", "fitness", "handicap", "prestige", "sentinel", "signal" ], "content": "Introduction\n\nThe issue of altruism is still a major question in evolution. Trivers (1985) defined altruism as an \"act that confers a benefit on someone at a cost to the other (the altruist).\" \"…cost is measured by a decrease in reproductive success\". Several theories have been posited to suggest that the altruist gains indirectly, among them are group-selection (including group augmentation), kin-selection and reciprocal-altruism. All these theories are based on the assumption that the altruistic act reduces the reproductive success of the altruist, and were developed in order to explain how altruism has persisted despite its supposed shortcomings. Zahavi (1977); Zahavi (1990); Zahavi (1995) and Zahavi & Zahavi (1997) contended that many altruistic acts can be explained as activities that signal the performer's claim to social prestige, and that increase rather than decrease the fitness of the altruist. Consequently, contestants would be expected to compete to perform the altruistic act. Such competition has already been shown in babblers for allofeeding and feeding at the nest by yearlings (Carlisle & Zahavi, 1986); for mobbing of raptors (Anava, 1992); in confrontations with neighbours (Berger, 2002) and in allofeeding among adults (Kalishov et al., 2005).\n\nGuarding, the activity of one or more individuals taking turns to watch over the group, is a very common phenomenon among group-living birds and animals (see Ridley et al., 2013 for references). Many observations have confirmed that the presence of a sentinel allows the group to forage in comfort (Hollén et al., 2008; Ben Mocha, 2013). However, that guarding helps the group does not explain why a particular individual may forego foraging or any other occupation, in order to guard.\n\nIn this paper we present data showing that the alpha and beta male babblers compete to perform the altruistic act of guarding. Other aspects of the phenomena of guarding are not discussed.\n\n\nMethods\n\nArabian babblers are thrush-size, group-living song birds (Zahavi, 1989; Zahavi, 1990; Zahavi & Zahavi, 1997). The study took place at the Shezaf Nature Reserve, near Hazeva Field Study Center, in the Rift Valley, 30 km south of the Dead Sea, Israel (coordinates: 30_46¢N, 35_14¢E). The site is an extreme desert, summers are hot and dry. Mean winter rainfall amounts to 35 mm, but it may be as low as a few millimeters in dry years. This babbler population has been studied since 1971 (Zahavi, 1989; Zahavi, 1990). Groups are composed of 2–20 individuals. The birds are tame, and observers can make close observations without disturbing them. The lineage and life history of most of the individuals is known from the time they were colour-banded as nestlings for individual recognition. The groups are resident and territorial. They maintain a strict age- and gender-dependent dominance hierarchy. Only the alpha male and female usually reproduce in the single nest, although sometimes more than one male may father the young and more than one female may lay in the common nest (Lundy et al., 1998).\n\nAll group members share in activities such as defending the territory against intruders, sentinel activities, mobbing, incubation, feeding and caring for the young. There is very little overt aggression, except among the very young. The birds spend most of the time in close proximity, and when not foraging often clump, play or allopreen.\n\nObservations were carried out from August 2003 to April 2004, following four years of serious drought, in which reproduction was reduced from 180 nestlings and fledglings, ringed in 1997, to the low number of about 30 ringed in each of the years 2000, 2001 and 2002. As a result the population was old and very stable (Zahavi, 1990), enabling us to select a large number of similar groups, each comprising two adult males (Alpha male, M1; beta male, M2) and one adult female (F1). Some of the groups included one or two independent youngsters fledged in 2003 (Table 1). The males were not related to the females and thus competed for reproduction, except in one group (MTE), in which the beta male was the son of the breeding female. In another group (HNC), there was acute competition between father and son culminating in eviction of the father leaving a group of two. Observation of this group ceased following the eviction. Competition over breeding was also intense in the SAL group, in which M2 was observed to copulate with the female and on several occasions interrupted the copulation of the dominant pair by following them (Perl, 1996).\n\nAge (years, on 1/1/2004).\n\n* > before the age denotes years since the individual joined the study population as an adult.\n\nAll the birds in this study were colour-banded for individual recognition and tame to the extent that they readily accepted food from the hand of the observer. None of the groups inhabited a territory bordering directly on agriculture.\n\nData on group composition, their ages and relatedness among the males are presented in Table 1.\n\na. Guarding. When the group is searching for food, or occupied in some other activity, one individual will stop feeding and climb onto a high place (e.g. a tree), inspecting the neighbourhood. It is usually easy to distinguish the sentinel from an individual that is in the tree for another reason, such as feeding, resting, auto-preening, etc. The height and extent of exposure of the sentinel depends on environmental conditions: in the darkness of dawn or during danger, it perches on the edge of the canopy. With increasing light it takes a more exposed perch, climbing to the top of the canopy, or often onto a dry branch devoid of leaves high above the canopy, from where it can see and be seen for a great distance. Many groups have a preferred guarding perch in their territory, highly exposed above the canopy (Figure 1). Sometimes the sentinel flies directly to its guard post, but more often it climbs the tree, scanning around, then climbs to a higher and more exposed position and sometimes flies off to another tree. There is usually only a single sentinel, but when a predator or an alien babbler is sighted the whole group may perch for a while on the top of the tree. At the end of the guarding bout the sentinel either flies down directly or moves slowly into the canopy. A new individual starts its guarding bout at variable intervals after the previous sentinel had left its post. However, about 15% of the guarding bouts are started by the replacement of an active sentinel see below.\n\nIn contrast to many other species (Bell et al., 2010; Gaston, 1977), adult Arabian babbler sentinels do not emit regular calls, except for “alarm calls”, or shouts when interacting with distant babblers (Sommer, 2012). Young sentinels, however, often produce soft sub-songs while they guard. These vocalizations attract attention to the youngster, advertising its activity to the group.\n\nWe do not know the proximate cause for a babbler to assume guarding and we do not discuss this issue here. However, we do know that the presence of foreign babblers in the territory increases guarding activity by the residents. The presence of an alien female in the territory greatly increases guarding by both the breeding female and the other females (Ben Mocha, 2013). Food availability too has a profound effect: on a rainy day little guarding was observed, but on the following day a maximum duration of almost 45 minutes/h was reached, probably due to the availability of a large number of insects that had drowned in the flooding (Anava et al., 2002).\n\nb. Replacements of sentinels. We observed several methods of sentinel replacement: it could be direct, with the replacer flying directly to the guarding bird, with or without some food item, and the previous sentinel then leaving its perch; or indirect, with the replacer taking up a guarding position on another perch. If the replacing bird was a subordinate, it often perched below the dominant or on another, usually lower, tree, and climbed to the final guarding position only after the dominant had vacated it. Eventually, mostly within one minute, one of the birds would abandon its position. In most cases, if the original sentinel did not leave its perch the second one retreated. The conflict between the two was often manifested by both of them nervously preening themselves.\n\nc. Allofeeding. Adult babblers sometimes allofeed (Kalishov et al., 2005). This often happens when the feeder seeks to replace the sentinel. We occasionally observed how a babbler that intended to replace another by allofeeding, first looked at the sentinel, and then started searching for a suitable food item. Upon finding one, it flew directly to the sentinel, fed it and usually replaced it. The sentinel either accepted the food (acceptance), sometimes crouching in a begging position (accepting like a fledgling), or rejected it. Finally it either did or did not leave its post. Often the sentinel, which may well be aware of the intention of the feeder - left its post before the feeder reached it. On rare occasions, when the sentinel did not accept the food item, the replacer (always a dominant) aggressively pushed the food into its beak. On several occasions a subordinate babbler that had just refused food offered by the dominant, immediately approached the observer to take a small crumb of bread, suggesting that despite being hungry it had refused to accept the \"gift\" from another babbler. Such interactions are not common but we have witnessed several over the years (Zahavi & Zahavi, 1997).\n\nd. Social phases. We have defined three social phases: a. non-breeding; b. courtship, beginning when sticks are collected and lasting until the last egg has been laid; and c. incubation and feeding the young until independence. Breeding cycles were often aborted for various reasons.\n\nEvery group was visited on average every 7–10 days. However, visits were not equally distributed – during mate guarding and egg laying a group could be visited daily until the first day of incubation, when frequency of visits usually dropped.\n\nObservations started with first light, usually before the babblers had left their night-roost tree. For the first 2–3 hours they were followed without any interference by the observer. The data presented in this paper were collected during that period. Following these observations the babblers were offered some bread tidbits and water. This was done in order to induce allopreening, which was the main subject of the study (Dattner, 2005). In one group (MTE) an entire day of observations without interference was conducted once a month. Of those days, only observations from the first three hours of the morning are included in the data presented here. We recorded the time of ascent and descent of the sentinel, its identity, the way by which it took up its perch, whether there was another sentinel at the time, whether it was replaced, the identities of the replacer and the replaced, type of replacement (direct or indirect or with allofeeding, acceptance or rejection of the food). The observations were written up on cards, noting the exact time and circumstances, and were later transferred to Excel spreadsheets. The social phase of the group in relation to the breeding cycle was also recorded. Table 2 presents the number of observation hours for each group, at the different social phases.\n\nFirst three hours of the morning.\n\nCourtship phase: from collection of sticks to the start of incubation.\n\nWeight was measured occasionally by other researchers who monitored the groups on alternate days, but not on our \"observation days\". The babblers were weighed in the morning as soon as they left the night roost. The birds were lured into mounting a scale (Moznei Shekel) by placing tiny tidbits of bread on it. The data presented in Table 3 are averages of several measurements taken over several months. A few individuals were not weighed because they either refused to mount the scale or were absent for some reason.\n\nAverages of several dates. See text for details\n\nThe data presented in this paper summarize a total of 637 h of observations in the mornings without interference by the researchers: 212 h in the non-breeding phase, 322 h in the courtship phase (179 h during nest building and 143 h during mate-guarding), and 103 h while incubating and feeding the young. When not otherwise noted, numbers are averages per hour of observation.\n\nStatistical analysis was carried out using an online calculator for non-parametric Wilcoxon signed-rank test to compare the behavior of the alpha and the beta males in the group (http.//www.socscistatistics.com/Default.aspx). Effect size calculations were also carried out using effect size calculators (http://www.polyu.edu.hk/mm/effectsizefaqs/calculator/calculator.html).\n\na. Daily activity. Foraging for food was the main activity in the morning. Later in the day, when the birds were partly satiated, other activities dominated, such as allopreening (Dattner, 2005), play (Pozis-Francois et al., 2004) and, mainly in summer, sleep. Activity in the afternoon was mixed. This pattern of activity is reflected in the number of guarding bouts during the day (Supplementary material 1). In the first three hours the number of bouts was maximal, between 8–9 bouts per group per hour. Later, the number fell below 5, and averaged less than one bout per hour in the middle of the day (and down to zero in the hot hours of the summer). Sentinel activity was resumed together with the other activities in the afternoon, but did not reach the same level as in the mornings.\n\nb. Guarding. The mean (±SE) duration in which a sentinel was present in the morning was 22.75±1.3 (n=10 groups) minutes per hour of observation, meaning that even in the morning, at the time of maximal sentinel activity, a sentinel was present only for about one-third of the time. In every group there was great daily variability in the amount of sentinel activity, ranging from a minimum of 6.5 minutes to a maximum of 50.5 minutes per hour of observation (Supplementary material 2).\n\nThe alpha male (M1) guarded more often and for longer periods than the two other adults (M2 and F1; Figure 2). These differences were highly significant both for the duration of guarding and number of bouts (p<0.01, W=0, n=10. Effect sizes (Es) for the differences in the duration of guarding and for the number of bouts between M1 and M2 were both large (see Dataset, Figure 2). In 80% of the 241 observation days the alpha male guarded more than any other individual in the group. Only on 20% of the days did the beta male or the female guard somewhat more than the alpha. This often happened when the latter was busy incubating (Figure 11). There were no consistent differences in the number of bouts or in the duration of guarding between a beta male and a female.\n\na. Duration of guarding (minutes/h) and b. number of guarding bouts/h by M1 and M2.\n\nAs soon as a subordinate male became dominant – following the eviction or disappearance of the alpha male – its guarding increased to the level of dominant (Figure 3). One could argue that the former M2 was now guarding against the former M1, which might still be present at the border of the territory. However, in group BOT1 the eviction of the M1 occurred early on in the observation period (with the group thus becoming BOT2), and the former M2 maintained a high rate of guarding until it was itself evicted. All other cases of eviction occurred towards the very end or after the end of the present study.\n\nc. Relationship of guarding to body mass is presented in Figure 4. It is clearly evident that within each category (M1, M2 and F1) there was no correlation between the babbler's weight and its guarding effort (R=0.12092, p=0.57354). In five of the seven groups for which the weight of both males was known M2 was heavier than M1 (Table 3) and in all of them M1 guarded much more than M2 (see Dataset, Figure 4). As soon as an M2 became dominant, its guarding increased to match that of the previous dominant (Figure 3), without a corresponding change in its weight.\n\nd. Replacements of the sentinel. Replacements of a sentinel occurred on average about once per hour of observation. Thirty-percent of the replacements (32%±1.57, n=14) occurred within 0–1 minutes after the original sentinel had started its guarding bout. When not interrupted, only about 10 % (±1.24 SE, n=14) of the bouts lasted less than 1 minute. There was a significant difference between interrupted and uninterrupted bouts (p<0.01, W=0, n=14, Es: large.) (Supplementary material 3).\n\nThe number of replacements/h as well as the percentage of guarding bouts starting with the replacement of an active sentinel by an M1was higher than those by an M2 (Figure 5). There was a significant difference in number of replacements by M1 over that of by M2 (p<0.01, W=0, n=8, Es large) and in percentage of bouts starting by the replacement of an existing sentinel (p<0.05, Es: large).\n\na. Number of replacements/h and b. percentage of guarding bouts that started by replacing an active sentinel.\n\nIn about 20% of its guarding bouts M1 started its session by replacing an active sentinel. M2 did so in 11% and an F1 in 18% of their guarding bouts respectively.\n\nFigure 6 and Figure 7 show which individual replaced which other individual. Figure 6 presents the total number of replacements and number of direct and indirect replacements of M2 or F1 by M1 and of M1 or F1 by M2. In over 50% of encounters M1 replaced M2 directly. M2 never replaced M1 directly but they did replace the females directly (see Dataset, Figure 6).\n\nIn Figure 7 replacements of the young are also shown. In this figure replacements are presented as a percentage of the guarding bouts of the babbler being replaced. This was done to compensate for the low number of bouts by the young. Only groups that included young birds are presented in this figure.\n\nThe figure clearly reveals that M1 replaced the sentinels much more often than M2. M1 also replaced its adult partners as sentinels much more often than it replaced the young inexperienced birds (p<0.05, W=0, n=5, Es: large). M2 rarely replaced M1 but it did replace females and young.\n\ne. Allofeeding. (Figure 8, Table 4). On average, allofeedings during replacements were observed about once per observation session. Data in Figure 8 clearly show that M1 replaced M2 with allofeedings significantly more than it replaced and allofed the young (p<0.05, W=0, N=5, Es: large), although the latter were surely more in need of it. It is also apparent that the youngsters allofed each other more than they received from the adults. We never observed an M2 allofeeding an M1, although they do, on rare occasions, as was observed in other studies (unpublished report). M2 allofed both the females (Table 4) and the young (Figure 8). M2, and to a lesser extent the females, sometimes received the food in a crouching position, like fledglings. However, the food was also sometimes refused (Table 4).\n\nn/h= interactions per hour.; crouch= accepting as a fledgling (percentage of interactions); refusals (percentage of interactions).\n\nf. Social phase. The breeding phase had a profound effect on all aspects of guarding, especially on that of the alpha male (Figure 9–Figure 11). The number of guarding bouts by M1 increased significantly during the courtship phase but dropped back as soon as incubation started (p<0.01, W=0, n=6, Es:large).\n\nGuarding (bouts/h) by M1 and by M2 during a. mate guarding and b. the first day of incubation, in groups BOK, NAV, POL, SAL and TMR.\n\nThe number of replacements of M2 by M1 as well as the number of allofeedings of M2 by M1 also increased significantly during mate-guarding and then declined at the start of incubation (p≤0.05, W=0, n=6; Figure 10). In some of the groups the M2 also slightly increased its guarding bouts (Figure 9) in the courtship phase but it did not drop during incubation and feeding of the young.\n\nDuring the first days of incubation the breeding-pair often monopolized incubation. On those days the guarding by M1 declined and those of M2 increased significantly in all groups (p<0.05 W=0; n=5; Figure 11).\n\nDifferent types of replacements: indirect, direct and with allofeedings can be visualized in this video clip: https://youtu.be/H_EdXAbRu2g\n\n\nDiscussion\n\nThe data provided in this paper demonstrate that the alpha and beta males compete to guard. In addition to the alpha bird guarding more than any other individual, it also replaces and thus interrupts the guarding by other birds, especially that of the beta male, which competes with it over reproduction. The alpha male replaced the guarding beta male more than it replaced the young inexperienced birds (Figure 7 and Figure 8). About one-third of guarding replacements took place less than one minute after a particular individual had mounted the guarding perch (Supplementary material 3). This indicates that the sentinel that had just started its guarding bout was not yet hungry or tired, unlike what was suggested by Bell et al. (2010) for the pied babbler.\n\nThe dominant often replaced its subordinate directly (Figure 6) by landing next to its perch, whereas the subordinate did not replace the alpha directly, but indirectly, by perching below it or taking a post on a lower tree, waiting for it to leave. The motivation of the beta male to replace the alpha was evident, for as soon as the latter had vacated its perch the beta male often climbed up to occupy the same perch. The alpha male also allofed the beta male during the replacement, sometimes forcing it to accept the gift, while the latter often reacted by fluttering its wings like a fledgling (Figure 8, Table 4). The fact that all these behaviours increased significantly during courtship, when competition over breeding was maximal, and then declined at the start of incubation, after paternity had been decided and competition over immediate paternity was over (Figure 9 and Figure 10), highlights the competitive basis underlying the act of guarding.\n\nIt should be stressed that most of the replacements were performed quite gently. The replacing bird often clumped with and sometimes allopreened the soon to be replaced sentinel for a few seconds or more before the latter left. However, when it refused to leave, it was sometimes pushed and even pecked at by the alpha male. As already noted, although overt aggression among adult males within the group is rare, aggressive replacements do occasionally occur.\n\nWright et al. (2001a); Wright et al. (2001b); Wright et al. (2001c)) contended that a direct relation exists between a babbler's mass (weight) and the extent of its guarding: \"Overall, body mass explained much of the variation in individual sentinel effort both within and between birds\", and \"…found relatively little evidence that individuals compete for the chance to act as sentinels\".\n\nOur data not only clearly contrast these statements, but some of the data presented in the figures of Wright et al., contradict their own statements. They obtained a positive regression of mass vs. guarding effort (Figure 4 of Wright et al., 2001b) due to the inclusion of guarding by young immature group members, which were both lighter in weight and guarded much less than the adults. Indeed, the upper part of their figure differs little from our Figure 4, and clearly shows that within each of the upper three social categories (M1, M2, and F1) there is no correlation between body mass and guarding. In another study (Wright et al., 2001c), the average body mass of the beta males was given as greater than that of the alpha males (Figure 3 of that study); but in eight out of the ten control days the sentinel effort by the alpha male was higher than that of the beta (their Figure 2, see also Figure 1 of Wright et al., 2001b). Five of our beta males were heavier than the alpha males in their groups (Table 3), but guarded much less than the alpha. In yet another paper, Wright et al. (2001a) state that \"Change-overs between sentinels rarely revealed any social context\"; but Figure 3 of the same study reveals large differences in change-overs between the alpha and beta males. Moreover, it has frequently been shown that supplementing the babblers with food greatly increased sentinel activity (Bell et al., 2010; Cordovi, 1988, unpublished report; Wright et al., 2001b), suggesting that food availability constitutes a constraint on guarding, especially in the desert. However, except for the case of artificial differential feeding of one particular individual for single days (Wright et al., 2001b), food availability for the group did not alter the relative extent of guarding by the different hierarchical categories.\n\nWright et al. (2001a) also suggested that in large groups the load of guarding per bird was reduced. This argument was based on the division of the total time when a sentinel was present, by the number of birds in the group. However, our data show that the time that a babbler spends guarding depends mainly on its place in the hierarchy and other social and ecological aspects (including food availability), rather than on the mean number of birds in the group. Guarding by the maturing young does extend the overall period in which a sentinel is present (Supplementary material 2), but it does not reduce the \"load\" of any particular individual.\n\nWright et al. (2001b) concluded that \"...sentinel behaviour in Arabian babblers appears entirely consistent with recent (Bedenkoff's) models of selfish state-dependent sentinel behaviour \". Bedenkoff (1997); Bedenkoff (2001) proposed a model suggesting that \"the sentinel position is the safest place to be… when no other group member is on guard\" If that were indeed the case, why replace that sentinel? In contrast, in a recent paper, Ridley et al. (2013) found that in the pied babbler the sentinel is at a greater risk of predation and further from cover than the foragers. Our observations (unpublished report) suggest that this is the case also for the Arabian babbler.\n\nWhy did the beta males guard much less than the alpha males? In our groups the beta males were all mature and not much younger than the alphas in their group (Table 1). Whenever they had the opportunity they guarded as much as the alphas. During the first days of incubation the breeding pair monopolize the incubation. Later on the beta male as well as the young may share in the incubation. In the few groups that were observed on the first day of incubation, the alpha male spent most of its time incubating rather than replacing the beta male. Consequently, the beta male increased its guarding almost to the average level of the alpha on other days (Figure 11). One could argue that M2 was compensating for the absence of M1 – however, the large day-to-day fluctuations in the total amount of guarding (Supplementary material 2) are not compatible with the notion that there is a certain daily \"quota\" of guarding. Indeed, in most cases, on days in which the alpha bird reduced its guarding - it still guarded more than any other individual in the group. The large increase in guarding by the beta male following its eviction of the alpha (Figure 3) also indicates that the beta male's previous lower extent of guarding was not due to incompetence, or laziness or lack of desire to guard. We suggest rather, that guarding by the beta male was restricted by the alpha male.\n\nMany observations have confirmed the fact that the presence of a sentinel allows the group to forage in comfort (Hollén et al., 2008 and references cited therein; Ben-Mocha, 2013). However, this does not explain why a particular individual will feel obliged to stop feeding, or any other activity, and stand guard over the group. Our calculations show that even in the morning, at the time of maximal guarding activity, a sentinel was present only for about one-third of the time. Why should a babbler choose frequently to replace an active sentinel rather than to start guarding when no other individual is guarding, and thus extend the overall time during which a sentinel is present? That the alpha male guards more than any other member of the group, and often replaces a subordinate as soon as the latter seeks to begin guarding, suggests that the primary concern of the alpha male is to demonstrate that it is the one that is performing the altruistic act, and that it can control the beta male and replace it whenever it wishes.\n\nAnava (1992) studying mobbing of raptors in Arabian babblers, observed a similar competition among the males as well as among the females. In most groups the alpha male participated more than any other individual in all aspects of mobbing, and interfered with the mobbing of other group members, mainly that of the beta male; except in one group in which there was a single male and three adult females that competed over breeding. In that group the alpha female participated in the mobbing and guarding more than the male and interfered in the mobbing by the other females.\n\nCompetition over altruistic acts was also described in babblers with regards to: allofeeding and feeding at the nest by yearlings (Carlisle & Zahavi, 1986); confrontations with neighbours (Berger, 2002); and allofeeding among adults (Kalishov et al., 2005). All these altruistic activities confer benefit on the group and require investment by their performers.\n\nCompetition makes sense if the contestants gain from winning. Any sentinel, not necessarily the alpha male, will satisfy the need of the group for a guard. If the reason for guarding is solely for the benefit to the group, why should a dominant not let a willing subordinate individual guard? We suggest (Zahavi, 1990; Zahavi & Zahavi, 1997) that by investing effort in guarding and by intervening in the guarding of its competitors, a babbler demonstrates and signals its general ability and its control over its competitors.\n\nGroup members are aware of the presence of the sentinel, because in its presence they can feed and move about with greater ease; they know who is guarding (Bell et al., 2010) and are also attentive to the replacements and how these replacements are carried out. By guarding and by replacing a competitor, a babbler thus retains or increases its own prestige and reduces that of the competitor. We have suggested previously (Zahavi, 1995; Zahavi, 2008) that this investment constitutes the handicap that proves the honesty of the claim to prestige.\n\nWhy should a babbler care so much about its prestige? High prestige provides the individual with a greater share in the group's resources. Prestige functions like an invisible \"peacock's tail\": it attracts collaborators and deters rivals. Zahavi & Zahavi (1997) suggested that whereas rank is stable, the prestige of an individual changes constantly. Every individual in the group, including the females and the very young, has and cares about its own prestige, especially in relation to those closest to it in the hierarchy.\n\nIn more general terms, because acting altruistically may confer prestige on the performing individual, altruism can substitute for other means of obtaining prestige, such as overt aggression or wasteful display. This is especially important in such closely-knit social groups as the Arabian babblers. Threats and aggression can easily turn into fights that may result in the killing or eviction of the loser. The loser, if it survives the fight, becomes a refugee, while the winner loses a partner in the defense against its neighbours.\n\nAn altruist gains directly from the investment of performing altruistic acts, and the competition increases the likelihood that there will usually be a willing candidate to take upon itself the duty of the sentinel. Members of a social group are attentive to such acts because they both benefit from them and gain information regarding the social relationships among other members of the group. This may explain why altruistic acts are so common among animals living in cooperative groups.\n\n\nEthics\n\nThe birds were banded and tamed under a permit from the Israel Nature and National Parks Protection Authority. No other permits were required.\n\n\nData availability\n\nF1000Research: Dataset 1. Source data for the statistical analyses together with effect-size calculations., 10.5256/f1000research.6739.d97781 (Datner et al., 2015).", "appendix": "Author contributions\n\n\n\nData presented in this paper were collected by Arnon Dattner and Carmel Bezner (Dattner, 2005). Arnon Dattner designed, collected and summarized the observations. More recently (2015) he produced the attached video clip. Avishag Zahavi wrote the manuscript and Amotz Zahavi supervised the thesis and was involved in every detail of the presentation.\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. Arnon Dattner's observation were part of his Msc thesis for the Tel-Aviv University. Carmel Besner was a volunteer.\n\n\nAcknowledgments\n\nWe are grateful to Carmel Bezner for assistance in the observations; Oded Keinan, Yitzhak Ben-Mocha, Mandy Ridley, Ally Harari, Arnon Lotem, Tirtza Zahavi and Naama Zahavi-Ely for critical reading and suggestions; to Arnon Lotem for suggestions on the statistical analysis and to Naomi Paz for editorial assistance.\n\n\nSupplementary materials\n\nSupplementary material 1. Guarding (bouts/h, per group) throughout the day. Figure and source data for MTE group.\n\nClick here to access the data.\n\nSupplementary material 2. Duration of guarding (min/h, in the first three hours of the morning) by different members of SAL group on observation days in February 2004 (breeding phase). Figure and source data for SAL group.\n\nClick here to access the data.\n\nSupplementary material 3. Brief bouts (0–1min). A. As a percentage of uninterrupted guarding bouts or B. Bouts replaced 0–1 minutes after the start of the bout, as a percentage of total replacements of the replaced babbler.\n\nClick here to access the data.\n\n\nReferences\n\nAnava A: The value of mobbing behavior for the individual babbler (Turdoides squamiceps) (in Hebrew). M.S. thesis, Ben Gurion University of the Negev, Beer-Sheva, 1992.\n\nAnava A, Kam M, Shkolnik A, et al.: Seasonal daily, daytime and night-time field metabolic rates in Arabian babblers (Turdoides squamiceps). J Exp Biol. 2002; 205(Pt 22): 3571–3575. PubMed Abstract\n\nBednekoff PA: Coordination of safe, selfish sentinels based on mutual benefits. Ann Zool Fennici. 2001; 38(2001): 5–14. Reference Source\n\nBednekoff PA: Mutualism among safe, selfish sentinels: a dynamic game. Am Nat. 1997; 150(3): 373–92. PubMed Abstract \| Publisher Full Text\n\nBell MBV, Radford AN, Smith RA, et al.: Bargaining babblers: vocal negotiation of cooperative behaviour in a social bird. Proc Biol Sci. 2010; 277(1698): 3223–3228. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBen Mocha Y: The functional and social role of sentinel behavior among the Arabian Babbler (Turdoides squamiceps). M.S. thesis. Tel-Aviv University (In Hebrew with English summary). 2013.\n\nBerger H: Interference and competition while attacking intruder in groups of Arabian Babblers (Turdoides squamiceps). M.S. thesis. Tel-Aviv University (In Hebrew with English summary). 2002.\n\nCarlisle TR, Zahavi A: Helping at the nest, allofeeding and social status in immature Arabian babblers. Behav Ecol Sociobiol. 1986; 18(5): 339–351. Publisher Full Text\n\nDattner A: Allopreening in the Arabian babbler. M.S. thesis. Tel-Aviv University (In Hebrew with English summary). 2005.\n\nDattner A, Zahavi A, Zahavi A: Dataset 1 in: Competition over guarding in the Arabian babbler (Turdoides squamiceps), a cooperative breeder. F1000Research. 2015. Data Source\n\nGaston AJ: Social behaviour within groups of Jungle Babblers (Turdoides striatus). Anim Behav. 1977; 25(4): 828–848. Publisher Full Text\n\nHollén LI, Bell MBV, Russell A, et al.: Calling by concluding sentinels: coordinating cooperation or revealing risk? PLoS One. 2011; 6(10): e25010. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKalishov A, Zahavi A, Zahavi A: Allofeeding in Arabian Babblers (Turdoides squamiceps). J Ornithol. 2005; 146(2): 141–150. Publisher Full Text\n\nLundy KJ, Parker PG, Zahavi A: Reproduction by subordinates in cooperatively breeding Arabian babblers is uncommon but predictable. Behav Ecol Sociobiol. 1998; 43(3): 173–180. Publisher Full Text\n\nPerl J: Competition for breeding between Arabian babbler males (In Hebrew). M.S. thesis. Tel-Aviv University. 1996.\n\nPozis-Francois O, Zahavi A, Zahavi A: Social play in Arabian Babblers. Behaviour. 2004; 141(4): 425–450. Publisher Full Text\n\nRidley AR, Raihani NJ, Bell MB: Experimental evidence that sentinel behaviour is affected by risk. Biol Lett. 2010; 6(4): 445–448. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSommer C: Alarm calling and sentinel behaviour in Arabian babblers. Bioacoustics. 2011; 20(3): 357–368. Publisher Full Text\n\nSturm RD, Kirk DE: First principles of discrete systems and digital signal processing. Reading, Mass.: Addison-Welsley Publishing Company. 1988. Reference Source\n\nTrivers RL: Social evolution. Menlo Park, California: The Benjamin/Cummings Publishing Company, Inc. 1985. Reference Source\n\nWright J, Berg E, de Kort SR, et al.: Cooperative sentinel behaviour in the Arabian babbler. Anim Behav. 2001a; 62(5): 973–979. Publisher Full Text\n\nWright J, Berg E, de Kort SR, et al.: Safe selfish sentinels in a cooperative bird. J Anim Ecol. 2001b; 70(6): 1070–1079. Publisher Full Text\n\nWright J, Maklakov AA, Khazin V: State dependent sentinels: an experimental study in the Arabian babbler. Proc Biol Sci. 2001c; 268(1469): 821–826. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZahavi A: Arabian Babbler. In: Newton, I. editor. Lifetime reproduction in birds. London: Academic Press. 1989; 253–276. Reference Source\n\nZahavi A: Arabian Babblers: The quest for social status in a cooperative breeder. In Stacey PB and Koenig WD editors. Cooperative breeding in birds. Long-term studies of ecology and behavior. Cambridge: Cambridge University press. 1990; 103–130. Publisher Full Text\n\nZahavi A: Altruism as a handicap - the limitations of kin selection and reciprocity. J Avian Biol. 1995; 26(1): 1–3. Publisher Full Text\n\nZahavi A, Zahavi A: The Handicap Principle. Oxford University Press. New-York. 1997; 286.\n\nZahavi A: The handicap principle and signaling in collaborative systems. In: D'Ettore P Hughes DP editors, Sociobiology of communication, an interdisciplinary perspective. Oxford University Press. 2008; 1–9. Publisher Full Text" }	[ { "id": "10220", "date": "07 Sep 2015", "name": "Uzi Motro", "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\nA very stimulating paper, looking at the vigilance conflict in a different, original way. The idea that taking on a social task (such as guarding the group) that inflicts an obvious burden on the performer, is not necessarily an altruistic act, has already been presented by Zahavi and their associates. Their assertion is that although such a behavior looks altruistic, indeed it is not – by accepting the burden, the performer reliably indicates its high quality, thus gaining a prestige which increases its reproductive success. The contribution of the present paper is in providing real data, which unequivocally confirm their handicap assertion. However, I have some concern with the statistical analysis of the data. The units of sampling are, obviously, the various groups. Thus, the observations on the alpha males, on the beta males and on the females do not represent independent samples, but constitute paired observations. The correct statistical tool should be a paired-observation comparison, such as a paired t-test. Moreover, Bonferroni-type adjustments for multiple comparisons are needed in some places. I took the liberty of performing paired t-tests on the source data (Dataset 1), and to my satisfaction, although the p-values have changed, the conclusions remain valid. And a small comment: In the Introduction, the authors claim that \"However, that guarding helps the group does not explain why a particular individual may forego foraging or any other occupation, in order to guard.\" There are several works that demonstrate how guarding can be evolutionarily stable in certain situations (see below some examples). The novelty in the present paper is that it can explain the competition over performing the act of guarding. Pulliam, H. R., Pyke, G. H. & Caraco, T. (1982). The scanning behaviour of juncos : a game-theoretical approach. Journal of Theoretical Biology 95: 89-103.Parker, G. A. & Hammerstein, P. (1985). Game theory and animal behaviour. In: Evolution: Essays in Honour of John Maynard Smith. (Greenwood, P. J., Harvey, P. & Slatkin, M., eds.) pp. 73-94. New York: Cambridge University Press.Motro, U. & Cohen, D. (1989). A note on vigilance behavior and stability against recognizable social parasites. Journal of Theoretical Biology 136:21-25.", "responses": [ { "c_id": "1757", "date": "04 Feb 2016", "name": "Amotz Zahavi", "role": "Author Response", "response": "~~The statistical analysis was corrected using Motro's calculations. See new version." } ] }, { "id": "10649", "date": "02 Oct 2015", "name": "Stephen Rothstein", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 reports observations of guarding in Arabian babblers in considerable detail and does an excellent job of relating various aspects of guarding to variables such as a bird’s dominance rank and its manner of replacing an individual who is already guarding. The results are new and important and the paper convincingly shows what looks like an element of competition in terms of who guards. This is a fascinating and valuable result which suggests that individuals that guard receive some increase in fitness from their guarding behavior. The paper argues that this fitness increment is via an increase in an individual’s prestige because time spent guarding is a handicap that takes away from time that could be spent feeding and because guarding may increase the guarding individual’s risk of predation. All of this train of logic holds together although I am not sure there is much actual evidence for the existence of prestige and its assumed effect on fitness. A problem as I see it is that there may be alternate explanations for the reason that dominant individuals seem to go out of their way to guard and even displace individuals that are already guarding. First, the alpha male may guard the most because he has the most to gain from guarding as he has fathered the offspring produced in the territory. Second, the assumption seems to be that guarding is done to reduce predation. But what if it is also done to repel conspecific intruders, especially males that might try and mate with the breeding female? The individual that would gain the most from repelling conspecific intruders is the alpha male and this could explain why alpha males do most of the guarding and even displace subordinate males from guarding as the latter would have less to gain from taking action against conspecific intruders. Guarding against intruding rival males explains why alpha males greatly increase guarding during the courtship because that is when extra-pair copulations would be most costly to their fitness. I did not see any clear explanation for this change in alpha males’ guarding under the prestige explanation. Guarding against intruding rival males could also explain why so many alpha male guarding sessions start with a direct displacement of another guarding bird as intruders are not as much of a threat (or any threat) to the fitness of other group members. Alpha males may sometimes allofeed birds already guarding because that may ease the transition and allofeeding may occur when an alpha males has found abundant food and is already satiated. Group members other than the alpha male may engage in guarding because they may eventually become the dominant male or female, i.e. they may benefit from maintaining the integrity of the group territory. If demonstrating to other group members a male’s ability to engage in seemingly altruistic acts is an important aspect of guarding, then alpha males should be more likely to initiate a guarding session when another group member is already guarding than when no one is guarding. The authors seem to have the data to test that prediction. The issue is admittedly complicated because there might be a greater need to guard against predators when no one else is guarding than when someone else is already guarding. But the relevant analysis might still be worth doing because it does have the potential to support the authors’ line of reasoning. There is no doubt that the babbler system is a very complicated one and that the Zahavis’ long term observations have made this into a classic system that has been very valuable to research on avian breeding and behavioral ecology in general. This paper adds to the novelty of the babbler system and it is only the final bit of the authors’ logic stream, that individuals incur a handicap so as to demonstrate their prowess and accrue prestige that remains unconvincing to me. More attention to alternative hypotheses and to testing these against the favored hypothesis might in the end provide more convincing support for the latter.", "responses": [ { "c_id": "1758", "date": "04 Feb 2016", "name": "Amotz Zahavi", "role": "Author Response", "response": "Q.---First, the alpha male may guard the most because he has the most to gain from guarding as he has fathered the offspring produced in the territory.Second, the assumption seems to be that guarding is done to reduce predation. But what if it is also done to repel conspecific intruders, especially males that might try and mate with the breeding female? The individual that would gain the most from repelling conspecific intruders is the alpha male and this could explain why alpha males do most of the guarding and even displace subordinate males from guarding as the latter would have less to gain from taking action against conspecific intrudersAnswer -There is no doubt that the detection of conspecifics is one of the most important aims of guarding. However, the idea that the subordinate male would have less to lose if the territory is taken over is incorrect, because when alien males or females take over the group in order to breed, all the members of the same sex as the alien in the original group (except sometimes the very young fledglings) are chased away (Zahavi A: (1990). Arabian Babblers: The quest for social status in a cooperative breeder. In Stacey PB and Koenig WD editors. Cooperative breeding in birds. Long-term studies of ecology and behavior. Cambridge: Cambridge University press. 1990; 103–130). The expelled individuals become refugees. It is therefore in the interest of every individual in the group to insure that intruders of the same sex will not take-over the group.Q .---Extra-pair copulations. The referee suggests that guarding against intruding rival males explains why alpha males greatly increase guarding during the courtship because that is when extra-pair copulations would be most costly to their fitness. \"I did not see any clear explanation for this change in alpha males’ guarding under the prestige explanation.\"Answer. Breeding females do not copulate with males of neighbouring groups, but may copulate with subordinate males of their own group that are not their offspring. Such extra-pair copulations are indeed of great concern for the alpha male. –Mate-guarding by the alpha male is aimed against copulations by the beta or lower ranking males that are not the descendants of the breeding female. This is why competition among same-sex members of the group increases during the breeding season. As there is almost no aggression among the same-sex adults, the competition is expressed mainly by increased guarding and by allofeeding – as demonstrations of dominance.Q.---Alpha males may sometimes allofeed birds already guarding because (1) that may ease the transition and (2) allofeeding may occur when an alpha males has found abundant food and is already satiated.Answer. (1) It has repeatedly been suggested that allofeeding is a demonstration of dominance (Kalishov A, Zahavi A, Zahavi A: (2005). Allofeeding in Arabian Babblers (Turdoides squamiceps). J Ornithol. 2005; 146(2): 141–150.; Kemp AC, Kemp MI (1980). The biology of the southern ground hornbill Bucorvus leadbeateri (Vigros). Ann Transvaal Museum 32:65–100.; Woolfenden GE, Fitzpatrick JW (1977). Dominance in the Florida scrub jay. Condor 79:1–12). As such it aggravates rather than mitigates the situation. This is manifested by the recipient often trying to avoid being fed (see \"refusals\" in Table 4).(2) In most cases the alpha male is not necessarily satiated when allofeeding. It often flies down to take tidbits from the observer immediately after taking up its guarding post following the allofeeding. Similarly, the individual that has just refused to accept food from its replacer will often readily accept the same kind of food when offered by the observer. Satiated individuals usually do not approach the offered food ." } ] }, { "id": "11476", "date": "09 Dec 2015", "name": "Raghavendra Gadagkar", "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 evolutionary origins and maintenance of altruism are indeed major unsolved problems in the study of animals and humans. Most people attempt to find ways in which altruistic behaviour, which is believed to be detrimental to the fitness of the actors, is nevertheless favoured by natural selection. Zahavi and his co-workers on the other hand, have consistently pursued the idea that so-called altruistic behaviour is actually beneficial to the actor. This is because the very fact that the altruist is willing (and capable) to undertake costly altruistic behaviour increases his prestige and therefore his access to mates. In this paper the authors attempt to provide support for their idea (often refereed to as the Handicap Principle), by showing that males of the Arabian babbler compete with each other to have the opportunity to undertake presumably costly, altruistic sentinel or guarding behaviour. Such a claim has been made by this group before but has been criticised by others who were either unable to find such pattern in sentinel behaviour of this species or argued that the observed pattern can be explained by other means. It may be very hard to conclusively demonstrate that males do or do not compete to perform sentinel behaviour or that all other explanations have been ruled out. The best way therefore to test the Handicap Principle in this case is to measure the actual costs and benefits of sentinel behaviour. This has not yet been done and is perhaps hard to do. Thus until such costs and benefits can be measured, we continue to rely on understanding and explaining the patterns of sentinel behaviour. In such a situation, wide acceptance of the Handicap Principle will necessarily depend on other researchers finding evidence in support of it. More research, more data and reiteration by the same authors and their associates are unlikely to be sufficient. I would therefore argue that this is treated as an open question and researchers finding evidence for and against the Handicap Principle should have the opportunity to publish their findings and their interpretations, as long as they are scientifically rigorous. In this spirit I support the dissemination of this paper but to make it scientifically rigorous I recommend that the authors revise their manuscript in response to my comments below. Introduction, last paragraph: If the authors wish to argue that altruistic acts are not actually altruistic, they should not continue to say “altruistic act of guarding”. Perhaps they should something like “supposedly altruistic act of guarding”. Under the heading “Descriptions of guarding patterns a. Guarding”, the authors state that: “It is usually easy to distinguish the sentinel from an individual that is in the tree for another reason, such as feeding, resting, auto-preening, etc.” The authors should say clearly how to make such a distinction between a sentinel and a bird that is in the tree for another reason, so that other researchers can try to make the same distinction and it does not remain only a tacit knowledge of the present authors. I first assumed that the few sentences that follow this describe how to make the distinction but I am not sure of that. If that is indeed so, the authors should be explicit about it. More generally speaking, many things that Zahavi and his co-workers ‘know’ is a kind of tacit knowledge to which outsiders have no access. For Zahavi’s ideas to become more widely acceptable, they will have to make the effort to describe the sources of their knowledge and the reason for their convictions more transparent and thus allow others to enter into the same knowledge space. In the same section at the end the authors mention that “but on the following day a maximum duration of almost 45 minutes/h was reached, probably due to the availability of a large number of insects that had drowned in the flooding (Anava et al, 2002)”. It is not clear why “high availability of insects” should increase guarding. Please explain. Under the heading “Descriptions of guarding patterns b. Replacement of sentinels”, the authors state in the last line: “The conflict between the two was often manifested by both of them nervously preening themselves”. How do we know there was conflict? Under the heading “Descriptions of guarding patterns c. Allofeeding”, the authors state in the end “… suggesting that despite being hungry it had refused to accept the “gift” from another babbler”. What is the interpretation of refusing to accept food despite being hungry, especially for a subordinate? Please elaborate. In Fig. 2, there is no mention of F1. In all the figures, the colour contrast between different bars is very poor.", "responses": [ { "c_id": "1756", "date": "04 Feb 2016", "name": "Amotz Zahavi", "role": "Author Response", "response": "1–Q. If the authors wish to argue that altruistic acts are not actually altruistic, they should not continue to say “altruistic act of guarding”. Perhaps they should something like “supposedly altruistic act of guarding”.Answer: Although altruism is defined by biologists as an act that helps others at a cost in fitness to the altruist, this is not the only definition of the term. Ordinary dictionaries define altruism as an investment that helps others. It is not dependent on whether or not the fitness of the altruist is reduced as a result, consequently, guarding when the rest of the group is feeding is indeed an altruistic act.We have repeatedly suggested that, on average, the direct fitness of the altruist is increased, and that \"This may explain why altruistic acts are so common among animals living in cooperative groups.\" See also: \"Altruism and moral behaviour.\" (page 149, in Zahavi A, Zahavi A: The Handicap Principle).In a more recent paper (~~Zahavi, A. and Zahavi, A. (2012). The logic of analog signaling and the theory of signal selection. Israel Journal of Ecology & Evolution. 58, 269-278) we suggest use of the term \"investment\" rather than \"cost\" because observers of altruistic acts can evaluate the investment of the altruist; whereas, when the investment is described as \"cost\" this already implies that it reduces the fitness of the altruist which is not necessarily the case2. Description of Guarding. Our \"questionable sentence\" is preceded by a description of sentinel activity. This description does not differ from that used in almost every study dealing with sentinel activity3. This is a short way of writing: \"Food availability has a profound effect: On a rainy day (for instance, Feb 22 in figure 2 , supplementary material) little guarding was observed - because it was difficult to find food while on the following day (for instance Feb 23, in the same figure) a maximum guarding duration of almost 45 minutes/h was reached, probably due to the availability of a large number of insects that had drowned in the flooding, facilitating spending less time on looking for food and more time on guarding\"4. See the attached video clip.5. If allofeeding may be considered an expression of dominance (Woolfenden and Fitzpatrick (1977) Kemp and Kemp (1980), Kalishov Zahavi and Zahavi, (2005)), then refusing to be fed reduces the prestige of the donor and increases that of the receiver. The fact that the recipient came to us to get food showed that it had not refused the food from the would-be donor because it was satiated." } ] } ]	1	https://f1000research.com/articles/4-618
https://f1000research.com/articles/4-638/v1	27 Aug 15	{ "type": "Clinical Practice Article", "title": "Double-Blinding and Bias in Medication and Cognitive-Behavioral Therapy Trials for Major Depressive Disorder", "authors": [ "Douglas Berger" ], "abstract": "While double-blinding is a crucial aspect of study design in an interventional clinical trial of medication for a disorder with subjective endpoints such as major depressive disorder, psychotherapy clinical trials, particularly cognitive-behavioral therapy trials, cannot be double-blinded. This paper highlights the evidence-based medicine problem of double-blinding in the outcome research of a psychotherapy and opines that psychotherapy clinical trials should be called, “partially-controlled clinical data” because they are not double-blinded. The implications for practice are, 1. For practitioners to be clear with patients the level of rigor to which interventions have been studied, 2. For authors of psychotherapy outcome studies to be clear that the problem in the inability to blind a psychotherapy trial severely restricts the validity of any conclusions that can be drawn, and 3. To petition National Health Insurance plans to use caution in approving interventions studied without double-blinded confirmatory trials as they may lead patients to avoid other treatments shown to be effective in double-blinded trials.", "keywords": [ "psychotherapy", "cognitive-behavioral therapy", "outcome studies", "blinding", "clinical trials" ], "content": "\n\nPsychotherapy clinical outcome trials for major depressive disorder (MDD) are often described as “randomized”, “controlled”, “single-blind”, etc. These words may not adequately describe the level of methodologic rigor of the design of a trial for MDD because the endpoints are subjective symptom ratings1, and the inability to double-blind MDD psychotherapy outcome trials is a crucial problem in the methodology of these trials2. Cognitive-behavioral therapy (CBT) is a widely-used type of psychotherapy in the treatment of MDD, however, CBT is very difficult if not impossible to double-blind because the subjects are actively involved in the therapy3. While clinical trials of CBT are often called “single-blind” because the raters are blind to treatment allocation, “single-blind” in a clinical trial is actually defined as a case where the subjects are blind, not the raters2,4.\n\nThe evaluation of MDD efficacy is more complex in some ways than that for objective endpoints of, say, tumor size, cholesterol level, or survival years. MDD may be diagnosed in a variety of persons, some with more psychological distress, and some with more neurovegetative symptoms. The symptoms of MDD are measured on rating scales whose scores become the endpoints of the study. All of the items on these rating scales are subjective, and some items like hopelessness and low self esteem are likely to improve with non-specific aspects of receiving care that include the hope and expectation inherent in belief in the treatment, compared with other symptoms such as lethargy and insomnia5.\n\nIt is well known that non-effective drugs and placebo pills will both show an average 30% improvement in depression scores from baseline, not just due to spontaneous improvement from waiting6. This “placebo effect” is thought to be due to hope and expectation of improvement on the part of the patient7.\n\nWe are also concerned that the term “evidence-based” is used in descriptions of the validity of a specific therapy without being clearly defined. While not foolproof, a double-blind design to control for expectations in antidepressant confirmatory studies is crucial in order to decrease potential bias2,8.\n\nNon-experimental comparative designs may also be used to make clinical inferences, however, this requires that studies include a number of conditions including: that the study subjects need to provide valid observations for the biological question under study; and the effect of the treatment must be large compared with random error and bias2. These conditions are extremely hard to meet in MDD where symptom reports are subjective1.\n\nWe think that using the term “evidence-based” for both double-blinded clinical drug trials as well as for unblinded psychotherapy trials confuses a consistent definition for “evidence-based”. We opine that the most valid definition of “evidence-based” is that of evidence garnered from the results of confirmatory trials of antidepressants that require double-blinding (http://cpnp.org/resource/mhc/2014/01/antidepressant-medications-fda-approval-process-and-need-updates)9. In this way, clinical trial designs with the strongest control level would be the standard for “evidence-based” data, although we acknowledge that double-blind clinical trials may also have various design and/or operational problems leading to invalid results.\n\nWe would like to illustrate how the combination of the placebo effect, along with the inability to double-blind a psychotherapy trial, can lead to bias in the results.\n\nFigure 1 illustrates the effect hope and expectation vs. pharmacological effect may have on improved depression scores. Ratings of depressive symptoms are subjective, some symptoms of which thus may be amenable to a subjective sense of improvement with the hope and/or expectation of entering a trial. Both subjects and ‘treaters’ are blind to the content of the pill received. Group B, is given a known antidepressant, but the subjects are blind to the nature of the pill. Blinding allows the study to show any unbiased antidepressant effect additional to hope and expectation.\n\nThe unblinded trial makes an unbiased evaluation of the efficacy of these pills impossible. Even more than just saying a study was “blinded”, however, absolute concealment of what treatment was allocated is crucial in order to avoid bias10. The study should clearly describe how they maintained the blind and employ an “exit analysis” to confirm that subjects were not aware of their treatment allocation.\n\nFigure 2 illustrates a non-blinded psychotherapy efficacy trial, using a “discussion” group and a “CBT” group. Because subjects are openly receiving the intervention given in a psychotherapy, it is essentially impossible to blind a psychotherapeutic intervention. Any type of psychotherapy could be used in this model, but CBT is an instructive case because there are workbooks and specific tasks given to subjects making it clear to subject that they are in the CBT group. The subjects and therapists are both told of the type of therapy received.\n\nGeneral Discussion refers to a non-directive, non-supportive discussion that is generally assumed to have no effect on MDD and represents a group that would have no expectation or hope of receiving a specific and directive therapy like CBT for the purposes of demonstrating the effect of unblinding for these groups. Assuming that “General Discussion” should not be effective in MDD, the discussion group’s improvement would then be similar to the placebo effect seen in a drug trial, and psychological placebos may also be as effective as accepted psychotherapies in MDD11.\n\nA third person who rates the degree of depression throughout the study should not know the therapy received (called a “masked rater”), but any bias on the part of the subject will just be part of the ratings reported by the subject to, and recorded by, the rater. The study is open so that it is not really possible to assess how much of each group’s improvement is due to the placebo effect, actual efficacy, or a bias towards CBT in knowing one is receiving CBT and/or hope from the act of actively trying to decrease negative thoughts as is done in CBT.\n\nThe importance of blinding in CBT interventions for psychiatric disorders was supported by a large meta analysis. Controlling for placebo and blindedness, a meta-analysis of data from published trials of CBT that showed CBT fared no better than non-specific control interventions in the treatment of schizophrenia and did not improve relapse rates, CBT showed no effect in prevention of bipolar disorder episodes, and only small treatment effects were seen in studies of MDD12.\n\nIn our location in Japan, the Japanese National Health Insurance (NHI) system added CBT as a reimbursable procedure for MDD in 201013. It is concerning to us for a National Health Insurance system to provide reimbursement for a treatment of MDD that does not have the same scientific rigor that a double-blind study of an antidepressant would have. This is similar to the NHI situation in the UK as discussed by Lynch12.\n\nThe suicide rate in Japan is among the highest in the world (http://www8.cao.go.jp/jisatsutaisaku/whitepaper/en/w-2013/pdf/chap1-1_p2-3.pdf, Accessed on December 4th, 2014.)14, and it is possible that treatments for MDD that do not have confirmatory double-blind clinical trials may lead patients with serious depression away from other approved treatments that do have this confirmation. The fact that the organizational relationship of the Ministry of Health, Labour and Welfare (MHLW) who funded the CBT studies also decides on the make up of the committees at an organization (called the Chuikyo) that determines approval for reimbursement by the NHI is also of concern (http://www.japantimes.co.jp/life/2006/03/14/lifestyle/who-is-paying-the-price-of-health-care/#.VIAGhMlWrkc, Accessed March 20th, 2015)15.\n\n\nConclusions\n\nThe conclusions of the rationale presented in this paper would be that for MDD:\n\n1. Pill placebos show considerable positive effect on disorders with subjective endpoints such as those used to rate MDD5–7.\n\n2. Psychological placebos may be as effective as accepted psychotherapies11.\n\n3. Psychotherapy clinical trials are non-blinded studies, and cannot effectively be double-blinded. Calling these studies “single-blind” obfuscates the non-blinded nature of these studies and is not in line with the definition of “single-blind” in a clinical trial4.\n\n4. It is imperative that any intervention for a disorder with subjective endpoints such as MDD requires the same rigor in double-blinding in order to conclude that the results show “efficacy” or are “evidence-based”. This paper proposes to use the term, “partially-controlled clinical data” in place of “evidence-based clinical data” for results obtained from unblinded studies.\n\nThe implications for practice are, 1. For practitioners to be clear with patients the level of rigor to which interventions have been studied, 2. For authors of psychotherapy outcome studies to be clear that the problem in the inability to blind a psychotherapy trial severely restricts the validity of any conclusions that can be drawn, and 3. To petition National Health Insurance Plans to use caution in approving interventions studied without double-blinded confirmatory trials as they may lead patients to avoid other treatments shown to be effective in double-blinded trials.\n\nThe limitations of this paper are that the lack of double-blinding does also not prove that the psychotherapy intervention is not helpful in some way to the indication being treated. Clinical opinion and consensus may guide how a psychotherapy will be used in practice.\n\nWe hope this paper can stimulate more research related to problems in blinding of psychotherapy outcome studies, the potential economic and clinical costs of providing or not-providing private or national health reimbursement for psychotherapeutic interventions, and further discussion on how our official professional organizations and national research centers will define “evidence-based” in relation to interventions for major depressive disorder.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed. The author has no financial interests, activities, relationships, and affiliations other than those affiliations listed in the title page of the manuscript. There was no data collected or analyzed for this paper.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe author thanks Mr. Burton Berger for help in preparation of the Figures originally done on Microsoft Excel.\n\n\nReferences\n\nWood L, Egger M, Gluud LL, et al.: Empirical evidence of bias in treatment effect estimates in controlled trials with different interventions and outcomes: meta-epidemiological study. BMJ. 2008; 336(7644): 601–605. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPiantadosi S: Clinical Trials: A Methodologic Perspective, 2nd ed. New York: Wiley-Interscience; 2005. Publisher Full Text\n\nBeck AT, Rush AJ, Shaw BF, et al.: Cognitive Therapy of Depression. New York, NY: Guilford Press; 1979. Reference Source\n\nFriedman LM, Furgerg CD, DeMets DL: Fundamentals of Clinical Trials, Third Edition. Springer; 1998. Reference Source\n\nCuijpers P, Driessen E, Hollon SD, et al.: The Efficacy of non-directive supportive therapy for adult depression: a meta-analysis. Clin Psychol Rev. 2012; 32(4): 280–291. PubMed Abstract \| Publisher Full Text\n\nMarchesi C, De Panfilis C, Matteo T, et al.: Is placebo useful in the treatment of major depression in clinical practice? Neuropsychiatr Dis Treat. 2013; 9: 915–920. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRutherford BR, Mori S, Sneed JR, et al.: Contribution of spontaneous improvement to placebo response in depression: a meta-analytic review. J Psychiatr Res. 2012; 46(6): 697–702. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNaudet F, Millet B, Reymann JM, et al.: Improving study design for antidepressant effectiveness assessment. Int J Methods Psychiatr Res. 2013; 22(3): 217–31. PubMed Abstract \| Publisher Full Text\n\nHanrahan C, New JP: Antidepressant Medications: The FDA-Approval Process and the Need for Updates. Ment Health Clin. 2014; 4(1): 11–16. Publisher Full Text\n\nSchulz KF, Grimes DA: Blinding in randomised trials: hiding who got what. Lancet. 2002; 359(9307): 696–700. PubMed Abstract \| Publisher Full Text\n\nWampold BE, Minami T, Tierney SC, et al.: The placebo is powerful: estimating placebo effects in medicine and psychotherapy from randomized clinical trials. J Clin Psychol. 2005; 61(7): 835–54. PubMed Abstract \| Publisher Full Text\n\nLynch D, Laws KR, McKenna PJ: Cognitive behavioural therapy for major psychiatric disorder: does it really work? A meta-analytical review of well-controlled trials. Psychol Med. 2010; 40(1): 9–24. PubMed Abstract \| Publisher Full Text\n\nOno Y, Furukawa TA, Shimizu E, et al.: Current status of research on cognitive therapy/cognitive behavior therapy in Japan. Psychiatry Clin Neurosci. 2011; 65(2): 121–129. PubMed Abstract \| Publisher Full Text\n\nCabinet Office, Government of Japan. White paper on Suicide Prevention in Japan. 2013. Reference Source\n\nOtake T: Who is paying the price of health care? Committee slow to bring transparency to matters of national insurance. The Japan Times. 2006. Reference Source" }	[ { "id": "10898", "date": "23 Oct 2015", "name": "Gordon Parker", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 straightforward paper, well written and taking the reader through the difficulties of undertaking and interpreting psychotherapies when no placebo control or equivalent control strategy is available and therefore worthy of indexing.", "responses": [] }, { "id": "11959", "date": "25 Jan 2016", "name": "Keith R Laws", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 a few comments on Dr Berger's manuscript, which raises interesting discussion pointsDr Berger states that “Psychotherapy clinical outcome trials for major depressive disorder (MDD) are often described as “randomized”, “controlled”, “singleblind”, etc. These words may not adequately describe the level of methodologic rigor of the design of a trial for MDD because the endpoints are subjective symptom ratings” (my italics).This latter point might benefit from some expanding and clarifying. All current psychiatric ratings are subjective to some extent (in pharma trials as well), but there are a variety of subjectives. For example, differences in terms of whether such 'subjective' assessment occurs using a self-rating tool (like the Beck Depression Inventory: BDI) or a clinician-rating scale (e.g. Hamilton Rating Scale for Depression). This is especially pertinent in depression trials as self-rating tools like BDI are frequently used and in effect, this renders trials 'open'.As Dr Berger states, meta-analyses show quite clearly that open psychotherapy trials can produce highly inflated effect sizes (see also Jauhar et al. 2014). The latter meta-analysis proves two points a) that compared with even single-blind trials, open trials of CBT can inflate effect sizes hugely and b) that contrary to what some say, psychotherapy trials can be very effectively blinded to assessors. I agree that the term ‘evidence-based’ in psychotherapy has been rendered somewhat meaningless and glosses over -what could be - important variability in terms of what this phrase covers. In some instances, paradoxically, the phrase is offered as an bland substitute for providing actual evidence...not unlike the often repeated “as recommended by NICE” - a shorthand way of avoiding the presentation and evaluation of actual evidence ... as if these phrases were chiselled in stone atop Mount Sinai. Study quality is undoubtedly the key factor and conflating evidence from double-blind, single-blind and open trials under the rubric of ‘evidence-based’ is unhelpful - especially to patients who require guidance on the efficacy of prospective psychotherapy as Dr Berger argues. Indeed, it would be much more helpful to patients and clinicians if bodies such as NICE incorporate study quality directly into their analysis of the evidence-base and hence, their recommendations. Its worth noting in passing that even when meta-analyses might reveal the same numerical effect size for a drug and a psychological intervention, this does not indicate equivalence of efficacy. Again, it depends crucially upon the quality of the data underpinning each effect size - so claims that CBT and anti-depressants have comparable effects really depends on the assumption that designs are equivalent (which they clearly are not). As Dr Berger suggests, drug trials are better controlled and so, any equivalence is more apparent than real. The proposal to use “partially-controlled clinical data” in place of “evidence-based clinical data” certainly highlights a key difference, though I guess it all depends upon the extent to which psychological therapy can be squeezed into the 'RCT template' that has served drug evaluation so well…RCTs of psych therapies are necessarily at best single blind or at worst (and still very often) ‘open’. Some psychotherapy advocates would of course make a special case for psychotherapy - that it is simply not amenable to the same paradigms as drug studies and perhaps Dr Berger could expand on how he views the validity of such counter-arguments.", "responses": [] } ]	1	https://f1000research.com/articles/4-638
https://f1000research.com/articles/5-133/v1	03 Feb 16	{ "type": "Review", "title": "Up in the air: Untethered Factors of Auxin Response", "authors": [ "Samantha K. Powers", "Lucia C. Strader", "Samantha K. Powers" ], "abstract": "As a prominent regulator of plant growth and development, the hormone auxin plays an essential role in controlling cell division and expansion. Auxin-responsive gene transcription is mediated through the TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX (TIR1/AFB) pathway. Roles for TIR1/AFB pathway components in auxin response are understood best, but additional factors implicated in auxin responses require more study. The function of these factors, including S-Phase Kinase-Associated Protein 2A (SKP2A), SMALL AUXIN UP RNAs (SAURs), INDOLE 3-BUTYRIC ACID RESPONSE5 (IBR5), and AUXIN BINDING PROTEIN1 (ABP1), has remained largely obscure. Recent advances have begun to clarify roles for these factors in auxin response while also raising additional questions to be answered.", "keywords": [ "auxin", "cell division", "gene" ], "content": "Introduction\n\nThe plant hormone auxin plays a vital role in nearly every aspect of plant growth and development1. Auxin-responsive gene expression relies on the TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX (TIR1/AFB) pathway to trigger the expression of genes controlling auxin-regulated cell division, expansion, and differentiation2,3. Whereas the role of the TIR1/AFB pathway in the auxin signal transduction pathway has been well established, the existence of additional components raises the possibility that we have yet to uncover the entire story of auxin signaling.\n\nAuxin signaling through the TIR1/AFB pathway involves three major protein families (Figure 1A) – the auxin-binding TIR1/AFB F-box proteins, the AUXIN RESPONSE FACTOR (ARF) transcription factors, and the AUXIN/INDOLE-3-ACETIC ACID INDUCIBLE (Aux/IAA) repressor proteins2,3. In the absence of auxin, the Aux/IAA proteins repress activity of the ARF transcription factors4. In the presence of auxin, the TIR1/AFB F-box proteins, which participate in a SCF (Skp1-Cullin-F-box) E3 ubiquitin ligase, interact with Aux/IAA repressor proteins to form a co-receptor, with auxin acting as the “molecular glue”4–6. This interaction results in ubiquitylation and consequent degradation of the Aux/IAA repressor proteins through the 26S proteasome, relieving repression of the ARF transcription factors and allowing for auxin-regulated gene transcription7. Interactions among these three protein families is now understood at a molecular level2 and provides a signal transduction pathway that controls auxin-responsive gene transcription in plants. For recent reviews of the TIR1/AFB pathway, please see 1,3.\n\n(A) Model of the TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX (TIR1/AFB) signaling pathway. Auxin promotes the formation of the TIR1/AFB Auxin/INDOLE-3-ACETIC ACID INDUCIBLE (Aux/IAA) co-receptor to promote the ubiquitylation and subsequent degradation of the Aux/IAA repressor. Aux/IAA degradation relieves repression of AUXIN RESPONSE FACTOR (ARF) transcription factors, allowing for auxin-responsive gene expression. One of the transcript families upregulated by auxin is the SAUR family. The small SMALL AUXIN UP RNA (SAUR) proteins encoded by these transcripts have been suggested to play roles in multiple processes, one of which is interaction with and inhibition of members of the PP2C.D family of phosphatases, which act to regulate H+-ATPase activity. Further, INDOLE-3-BUTYRIC ACID RESPONSE5 (IBR5) and MITOGEN-ACTIVATED PROTEIN KINASE12 (MPK12) have been implicated in regulating auxin-responsive gene transcription; this regulation is not through destabilization of the Aux/IAA repressors, suggesting a yet-to-be discovered mechanism of regulating auxin-responsive gene expression. For in-depth reviews of the TIR/AFB signaling pathway, please refer to 1,3. For an in-depth review of SAUR proteins, please refer to 15.\n\n(B) Model of the S-PHASE KINASE ASSOCIATED PROTEIN 2A (SKP2A) signaling pathway. E2FC/DPB repress expression of cell cycle genes. The F-box protein SKP2A binds auxin and promotes degradation of E2FC/DPB in an auxin-dependent manner. Degradation of E2FC/DPB relieves repression of cell cycle genes and allows for binding by activating E2F+/DP+ complexes. For an in-depth review of the SKP2A signaling pathway, please refer to 8.\n\n(C) Model of the putative AUXIN BINDING PROTEIN1 (ABP1) signaling pathway. Apoplastic auxin is bound by ABP1, which allows for interaction with the TRANSMEMBRANE KINASE RECEPTOR (TMK) family of leucine-rich repeat receptor-like kinases. ABP1 binding of auxin regulates RHO-LIKE GTPASE 2 (ROP2) and ROP6 activation and binding to ROP interactive CRIB motif-containing proteins (RICs) proteins to positively regulate microtubule polymerization and F-actin polymerization and also alter PINFORMED1 protein localization to alter auxin efflux. For an in-depth review of the ROP/RIC system, please refer to 54.\n\nIn addition to components of the TIR1/AFB pathway, other factors implicated in auxin response have been identified – however, roles for these components in regulating auxin signaling are much less understood. Although insight into the function of some of these factors, such as S-PHASE KINASE-ASSOCIATED PROTEIN 2A (SKP2A), SMALL AUXIN UP RNAs (SAURs), INDOLE 3-BUTYRIC ACID RESPONSE5 (IBR5), and AUXIN BINDING PROTEIN1 (ABP1), has been elusive, roles for these factors in regulating auxin outputs are slowly starting to be determined. In this commentary, we explore recent advances in our understanding of these factors and their roles in auxin response.\n\n\nSKP2A\n\nWhereas extensive work has gone into understanding the auxin-binding capabilities of TIR1 and its downstream effects on auxin-regulated developmental responses (Figure 1A), less is known about the molecular mechanisms that directly connect auxin to its role in cell division. Several cell cycle genes are upregulated by auxin8 and other cell cycle genes contain auxin response elements in their promoter regions; however, these do not appear to be upregulated by auxin8, suggesting that additional mechanisms linking auxin to cell division control may exist. Heterologously expressed SKP2A, an F-box protein involved in regulating the proteolysis of cell-cycle-related transcription factors9,10, directly binds auxin11 and may provide a mechanism for auxin-mediated regulation of cell division (Figure 1B).\n\nThe retinoblastoma-E2F pathway regulates the cell cycle through the interaction of E2F proteins with dimerization proteins (DPs) to form transcription factors that either activate or repress the expression of genes involved in cell cycle progression12. SKP2A incorporates into an SCF complex9,13 with E3 ubiquitin ligase activity13. Intriguingly, binding of auxin by SKP2A is necessary for degradation of E2FC and its dimerization partner DPB through the 26S proteasome11. Degradation of E2FC and DPB relieves repression of cell cycle control genes to allow cell cycle progression9,10. In addition to regulating the degradation of E2FC/DPB, auxin binding promotes proteolysis of SKP2A itself13, perhaps setting up a system in which auxin prevents SKP2A overfunction11. The skp2a mutant hyperaccumulates E2FC and DPB protein10,13. Further, expressing a SKP2A variant unable to bind auxin in the mutant background fails to rescue this phenotype, suggesting that auxin binding by SKP2A is required for E2FC/DPB degradation11. Overexpression of SKP2A results in increased cell division and induces lateral root primordia (LRP) formation, a process known to be dependent on auxin signaling9. The molecular phenotypes of the skp2a mutant combined with the phenotypes of the SKP2A overexpression lines suggest a role for SKP2A in promoting auxin-regulated cell division.\n\nInvolvement of SKP2A in auxin binding and consequent degradation of the cell-cycle regulators E2FC and DPB implicate this F-box as a missing link connecting auxin regulation to cell division; however, relatively little is known about this pathway. Many questions remain for SKP2A roles in auxin-regulated cell division control. For example, SKP2A directly binds auxin and functions as part of an SCF complex responsible for targeting downstream components for degradation – are additional factors, other than E2FC and DPB, targeted for degradation by SKP2A to contribute to auxin response? Further, are activating E2F+/EP+ complexes similarly regulated by the proteasome? What is the effect on plant growth and development if this auxin-induced degradation of these factors is disrupted? There appear to be no gross effects on plant morphology in the skp2a mutant; is this because of redundancy or is it indicative of a minor role for SKP2A in plant growth and development? Answering these questions will inform our understanding of SKP2A roles in mediating auxin effects on cell division.\n\n\nSAUR proteins\n\nAuxin regulation downstream of the TIR1/AFB pathway involves the induction of the early auxin response gene family SAURs. Initially discovered as auxin-induced transcripts in elongating soybean hypocotyls using a hybridization screen14, multiple lines of evidence have been used to assign SAUR functions in auxin-related aspects of plant growth and development, including cell expansion, tropic growth, and apical hook development15. Although SAURs represent the largest family of early auxin response genes, SAUR function in mediating auxin effects has only recently begun to be elucidated.\n\nCa2+ is a well-known secondary messenger regulating developmental and physiological aspects of plant growth16. Auxin has been proposed as a Ca2+ activating signal17; however, mechanisms connecting Ca2+ to auxin signaling have remained largely elusive. SAUR proteins from multiple species interact with calmodulin (CaM) in a calcium-dependent manner18–20 and additional SAUR proteins are predicted to contain a CaM-binding site15. Binding of SAUR70 to CaM or CaM-like proteins has been confirmed in planta19; however, further studies will be necessary to determine the extent and functional relevance of SAUR-CaM interactions in auxin-regulated calcium signaling.\n\nSeveral SAUR subfamilies, including SAUR19-24, are likely involved in cell expansion21; however, mechanistic insight into SAUR roles in this auxin-regulated response has been lacking. Auxin has long been proposed to induce cell elongation through an acid growth mechanism22, in which auxin is responsible for activating H+-ATPases to acidify the extracellular matrix, in turn activating expansins and promoting solute and water uptake to drive cell expansion. Recently, Spartz et al.23 determined that SAUR proteins promote phosphorylation of the C-terminal autoinhibitory domain of PM H+-ATPases, causing activation. In addition, several SAUR proteins interact with a group of the D-clade PP2C phosphatases to inhibit phosphatase activity23,24. These phosphatases likely modulate the phosphorylation status of H+-ATPases23. Thus, SAURs likely act to inhibit the PP2C.D inhibitors of H+-ATPase activity, suggesting a role for the SAUR proteins as positive effectors in auxin-mediated cell expansion through regulation of the PM H+-ATPase activity. If PP2C.D deactivates H+-ATPase activity through de-phosphorylation, it then follows that a kinase is necessary to activate H+-ATPases. Many protein kinases have been proposed to regulate H+-ATPase activity25 – perhaps one of these functions in an auxin-dependent manner to regulate H+-ATPases. Further, the identified SAUR-CaM interactions combined with the recently identified roles for SAUR proteins in regulating phosphatases raise the possibility that SAUR proteins could act as a link between calcium signaling, auxin, and phosphatase activity.\n\nRecent studies have provided increasing evidence of SAURs’ importance in auxin-regulated plant growth and development. The large families of these proteins identified in widespread plant species suggest multiple and diverse SAUR functions in auxin response. One possible mechanism for SAUR regulation of such varied auxin-related plant responses may be provided by combinatorial diversity in SAUR-PP2C.D interactions. In Arabidopsis, there are 81 SAURs26 and nine PP2C.D family members24, many of which have been proposed to have differential expression patterns throughout the plant27. Varying SAUR-PP2C.D combinations may regulate the phosphorylation status of distinct downstream elements to regulate different aspects of auxin response15. Interaction experiments using the different SAUR-PP2C.D combinations may uncover whether SAUR proteins from additional clades also interact with these phosphatases. Further, the distinct subcellular localization and various developmental processes associated with individual SAUR proteins15 suggest that SAUR targets in addition to H+-ATPases likely exist. Whereas recent studies have finally begun to illuminate SAUR molecular functions in auxin response, further research will surely uncover additional mechanisms connecting the SAUR proteins to auxin signaling.\n\n\nIBR5 and MPK12\n\nAuxin pathway roles for IBR5 and its interacting MAP kinase MPK12 remain enigmatic. The ibr5 mutant was initially isolated for its resistance to the auxin precursor indole-3-butyric acid (IBA)28, but subsequent studies revealed that ibr5 was resistant to all tested auxins29 and auxin transport inhibitors30. In addition to reduced physiological responses to exogenous auxin application, ibr5 mutants display developmental phenotypes29,31,32 and reduced auxin-responsive transcription29–32, suggesting roles for IBR5 in the auxin signaling pathway.\n\nIBR5 encodes a dual-specificity protein phosphatase29; related phosphatases de-phosphorylate MAP kinases33,34. Dual-specificity protein phosphatases are distinct from the PP2C-type phosphatases associated with SAUR activity (see above). IBR5 splice variants appear to play distinct roles in regulating plant growth and auxin responses and at least some of these roles may be independent of its catalytic activity31. However, the IBR5 catalytic cysteine is necessary for auxin responsive inhibition of root elongation30,31 and IBR5 can de-phosphorylate the Arabidopsis MPK12 in vitro35. Further, RNAi lines of MPK12 display auxin resistance35, suggesting that IBR5 and MPK12 play opposing roles in regulating auxin responsiveness.\n\nibr5 double mutants with other mutants that dampen auxin responses, including tir1, axr1, and aux1, exhibit additive auxin resistance in one or more bioassays30. Most notably, combining ibr5 with an auxin receptor mutant, tir1, greatly enhances auxin resistance relative to either parent30, consistent with the possibility that IBR5 effects on auxin response are TIR1 independent. Similar to other auxin-resistant mutants, ibr5 exhibits decreased levels of auxin-responsive transcripts29–32. However, unlike other characterized auxin-response mutants, Aux/IAA proteins are not stabilized in ibr5 and are actually destabilized30,31, again suggesting that IBR5 modulates auxin signaling in a manner unique from other known auxin response regulators. ARF proteins mediate auxin-responsive gene transcription and the primary known mechanism of ARF regulation is repression by Aux/IAA proteins3. The decreased auxin-responsive gene transcription29–32 combined with destabilized Aux/IAA repressor proteins30,31 observed in ibr5 mutants suggest that either Aux/IAA destabilization is not the sole mechanism of regulating ARF activity or that there exists an additional auxin signaling pathway that ends in regulating the same gene targets as the TIR1/AFB pathway. Elucidating IBR5 and MPK12 targets may help differentiate between these possibilities. Recently, IBR5 was found to interact with SUPPRESSOR OF G2 ALLELE SKP1 (SGT1b), HEAT SHOCK PROTEIN90 (HSP90), and the Toll/interleukin-1 receptor domains of CHILLING SENSITIVE 3 (CHS3), SUPPRESSOR OF NPR1-1 (SNC1), and RESISTANT TO P. SYRINGAE 4 (RPS4)36. IBR5 interaction with SGT1b may provide a mechanism for IBR5 regulation of auxin responses; mutants defective in SGT1b/ETA3 have been isolated as enhancers of tir1 auxin resistance, perhaps by modulating 26S proteasome activity37. SGT1b is a co-chaperone with HSP90. Further, TIR1 has recently been identified as a HSP90 client and HSP90 plays roles in integrating temperature and auxin signaling38; perhaps IBR5 is an additional HSP90 client to allow temperature and auxin response integration.\n\n\nABP1 and TMK1\n\nOpinions about ABP1, first discovered over 40 years ago, have varied over the years. ABP1 has been proposed to act as an apoplastic auxin receptor whose downstream signal transduction pathway regulates cytoskeletal rearrangement and internalization of auxin transporters in response to auxin39–41. Study of roles for ABP1 in plant auxin signaling was limited by the reported embryo lethality of null abp1 alleles42–45. In the absence of a viable allele, the field made progress in understanding ABP1 function by use of ABP1 knockdown lines, provided by expression of an inducible ABP1 antisense transcript or of an inducible single-chain fragment variable from a monoclonal antibody raised to ABP146. The identification of an EMS-generated TILLING line, abp1-5, carrying a point mutation in the auxin-binding pocket of ABP147,48 allowed for intense study of ABP1 functions in auxin signaling and spurred an explosion of ABP1-related discoveries, uncovering roles for this elusive auxin-binding protein in a wide variety of processes throughout plant development39. These new findings allowed for widespread acceptance of ABP1 as a bona fide auxin receptor41.\n\nThe recognition of ABP1 as an auxin receptor, however, has once again been called into question. A recent report showing that new abp1 null alleles display no obvious auxin-related or developmental phenotypes49 was contradictory to earlier reports of the embryo lethality of the abp1-1 null allele42–45 and the physiological and molecular phenotypes displayed by conditional knockdown and abp1-5 alleles39. This conflict was recently partially reconciled by the discovery that the embryo lethal phenotypes of abp1-1 and abp1-1s were caused by a loss of the neighboring BELAYA SMERT gene rather than from loss of ABP150,51 and that the abp1-1 allele, which is null for ABP1, displays no obvious morphological phenotypes when the BSM defect is rescued in the mutant50. Further, at least some of the phenotypes observed in the abp1-5 TILLING allele may be the result of a background mutation in PHYTOCHROME B52. Future work with new ABP1 genetic resources49,51 will be necessary to clarify the role of ABP1 in auxin signaling and plant development and will determine whether abp1 null alleles display molecular phenotypes.\n\nThe auxin-binding affinity of ABP1 combined with its widespread conservation throughout plants would suggest an important role for ABP1; however, lack of physiological phenotypes in the null mutants49,50 would suggest that ABP1 does not play a prominent role in Arabidopsis development. At this time, auxin-related roles for downstream components in the ABP1 pathway (Figure 1C) including the TRANSMEMBRANE KINASE (TMK) family of receptor-like kinases53 and the ROP-RIC system54 remain unchallenged. This is a turbulent time in the ABP1 field as new discoveries are being made and roles (or lack thereof) for this signaling pathway in plant growth and development are being clarified. Roles for ABP1 in auxin response and development have once again become controversial; clearly more work will be needed to reconcile conflicting reports in this area.\n\n\nConclusions and future directions\n\nThe existence of factors in addition to the components of the well-established TIR1/AFB pathway suggests that we have yet to uncover the entire story of auxin response. Recent advances in the field are slowly bringing to light roles in auxin signaling for each of these factors − SKP2A, the SAUR proteins, IBR5, and ABP1 – however, many questions still remain. In addition, although the TIR/AFB pathway appears to be well characterized, new structural data suggest that there are additional regulatory aspects of this pathway, including ARF proteins acting as molecular DNA calipers and ARF and Aux/IAA protein multimerization2,55 that have yet to be fully explored. Further research will undoubtedly uncover new regulatory mechanisms for the TIR/AFB pathway and molecular roles for these untethered factors in auxin signaling − the sky is the limit!\n\n\nAbbreviations\n\nABP1, AUXIN BINDING PROTEIN1; AFB, AUXIN SIGNALING F-BOX; ARF, AUXIN RESPONSE FACTOR; Aux/IAA, Auxin/INDOLE-3-ACETIC ACID INDUCIBLE; bHLH, basic Helix-Loop-Helix; CaM, Calmodulin; DP, Dimerization Protein; IBR5, INDOLE-3-BUTYRIC ACID RESPONSE5; MPK12, MITOGEN-ACTIVATED PROTEIN KINASE12; ROP, RHO-LIKE GTPASE; SAUR, SMALL AUXIN UP RNA; SCF, Skp1-Cullin-F-box; SKP2A, S-PHASE KINASE ASSOCIATED PROTEIN 2A; TIR1, TRANSPORT INHIBITOR RESPONSE1; TMK, TRANSMEMBRANE KINASE RECEPTOR.", "appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThis research was supported by the William H. Danforth Plant Science Fellowship Program (to S.K.P.), the National Science Foundation (IOS-1453750 to L.C.S.) the National Institutes of Health (R01 GM112898-01 to L.C.S.).\n\n\nAcknowledgements\n\nWe are grateful to Tara Enders, Elizabeth Frick, and Marta Michniewicz-Paciorek for critical comments on the manuscript.\n\n\nReferences\n\nEnders TA, Strader LC: Auxin activity: Past, present, and future. Am J Bot. 2015; 102(2): 180–196. PubMed Abstract \| Publisher Full Text\n\nKorasick DA, Jez JM, Strader LC: Refining the nuclear auxin response pathway through structural biology. Curr Opin Plant Biol. 2015; 27: 22–28. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang R, Estelle M: Diversity and specificity: auxin perception and signaling through the TIR1/AFB pathway. Curr Opin Plant Biol. 2014; 21: 51–58. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChapman EJ, Estelle M: Mechanism of auxin-regulated gene expression in plants. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDinesh DC, Villalobos LI, Abel S: Structural Biology of Nuclear Auxin Action. Trends Plant Sci. 2015; pii: S1360-1385(15)00278-2. PubMed Abstract \| Publisher Full Text" }	[ { "id": "12253", "date": "03 Feb 2016", "name": "Nihal Dharmasiri", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12254", "date": "03 Feb 2016", "name": "Dolf Weijers", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12255", "date": "03 Feb 2016", "name": "Richard M. Napier", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12256", "date": "03 Feb 2016", "name": "Mark Estelle", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12257", "date": "03 Feb 2016", "name": "Juan Carlos del Pozo", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-133
https://f1000research.com/articles/5-131/v1	03 Feb 16	{ "type": "Review", "title": "Recent advances in understanding hepatitis C", "authors": [ "Florian Douam", "Qiang Ding", "Alexander Ploss", "Florian Douam", "Qiang Ding" ], "abstract": "The past decade has seen tremendous progress in understanding hepatitis C virus (HCV) biology and its related disease, hepatitis C. Major advances in characterizing viral replication have led to the development of direct-acting anti-viral therapies that have considerably improved patient treatment outcome and can even cure chronic infection. However, the high cost of these treatments, their low barrier to viral resistance, and their inability to prevent HCV-induced liver cancer, along with the absence of an effective HCV vaccine, all underscore the need for continued efforts to understand the biology of this virus. Moreover, beyond informing therapies, enhanced knowledge of HCV biology is itself extremely valuable for understanding the biology of related viruses, such as dengue virus, which is becoming a growing global health concern. Major advances have been realized over the last few years in HCV biology and pathogenesis, such as the discovery of the envelope glycoprotein E2 core structure, the generation of the first mouse model with inheritable susceptibility to HCV, and the characterization of virus-host interactions that regulate viral replication or innate immunity. Here, we review the recent findings that have significantly advanced our understanding of HCV and highlight the major challenges that remain.", "keywords": [ "hepatitis C", "HCV", "Anti-viral therapy", "pathogenesis", "immune response" ], "content": "Introduction\n\nHepatitis C virus (HCV) is a single-stranded, positive-sense RNA virus of the Flaviviridae family. Although the inability to culture primary HCV isolates in vitro seriously hampered HCV research for the 15 years following its isolation in 19891, the generation of relevant in vitro systems in the early 2000s offered the first opportunities to accurately characterize the HCV life cycle. Since then, considerable progress has been made to understand HCV biology through the generation of increasingly relevant cell culture systems and animal models. These advances recently reached a milestone by the generation of effective direct-acting anti-viral agents (DAAs)2 able to cure HCV.\n\nHowever, a large number of challenges remain to significantly decrease HCV spread on a global scale. Indeed, 130 to 150 million people worldwide are still chronically infected with HCV (World Health Organization), and treatments remain poorly accessible because of their high costs. Moreover, their low barrier to resistance-associated mutations combined with their inability to treat challenging patient groups, HCV-induced liver disease, and hepatocellular carcinoma (HCC) all underscore the need for novel, cost-effective DAAs3–6. Finally, a prophylactic or preventive HCV vaccine is still urgently needed to significantly impact HCV spread worldwide.\n\nOverall, these challenges strongly highlight that our understanding of HCV and its related disease remains incomplete and that efforts need to be maintained to expand it. Here, we review the recent advances that have greatly contributed to improving our knowledge of hepatitis C and HCV and highlight the fundamental and clinical challenges that still need to be faced by the HCV scientific community.\n\n\nA. Hepatitis C virus life cycle\n\nHCV enters into hepatocytes through a dynamic, multi-step process involving multiple cell host factors7. As the primary attachment of viral particles at the hepatocyte surface occurs through interactions with lipoprotein receptors, the association between HCV particles and lipoproteins is critical for initiating the first step of virus entry8. After primary attachment, HCV particles interact with the tetraspanin CD81 via the viral E2 glycoprotein9, which, along with E1, constitutes a heterodimer complex at the surface of viral particles7. E2 interaction with CD81 is thought to induce signaling pathways involving epidermal growth factor receptor (EGFR)10,11 and HRas12 that together lead to the clustering of CD81-viral particle complexes with the tight junction protein claudin-1 (CLDN1)13,14. Although direct interaction between HCV particles and CLDN1 was not initially demonstrated, recent evidence supports such interactions15,16. Another tight junction protein, occludin (OCLN), is also critical for a late step of virus entry17,18, although its precise role during this process has not been clearly defined. CD81-CLDN1 clustering is thought to induce the internalization of viral particles through clathrin-dependent endocytosis19,20. The fusion of the viral particle and late endosome membranes, which is thought to be mediated by structural rearrangements of the E1E2 heterodimer complex, then results in the release of viral RNA into the cytosol.\n\nUncovering the spatio-temporal dynamics of hepatitis C virus entry. A considerable challenge in understanding HCV entry is accurately capturing the spatio-temporal dynamics of this process, as it involves a considerable number of host factors and regulators, both extracellularly and intracellularly. The recent identification of additional factors important for virus entry has only complicated this problem. Among those additional factors are the Niemann-Pick C1-like 1 (NPC1L1) receptor21, the transferrin-1 receptor22, and even more recently, via a proteomic approach, the serum response factor-binding protein 1 (SRFBP1)23. Although these new factors are thought to be important for late steps of virus entry, for rearrangement of lipoproteins within the viral particle, or for CD81-induced signaling pathways, their role in the spatio-temporal dynamics of virus entry still needs to be clearly defined.\n\nThe elusive hepatitis C virus fusion mechanism. In addition to viral entry dynamics, the HCV fusion process remains not fully understood because of the absence of crystal structures of E1 and the E1E2 protein complex. It was previously thought that E2 glycoproteins might harbor a class II fusion protein structure24, thus mediating the fusion between the viral and the endosomal membranes in a manner similar to flaviviruses. However, very recently, the structural resolution of the central core of the E2 protein25,26, E2core, revealed that E2 harbors a globular, non-extended structure that does not display any features of a class II fusion protein. In parallel, this finding has been strengthened by evidence that E1 might function as a fusion protein27–29, despite unusual N-terminal structural organization30. In the future, structural resolution of E1 and E1E2 complexes at pre- and post-fusion conformational states should unveil critical features of the fusion mechanism. Overall, this process would likely be unique in the Flaviviridae family and be mediated by a very original, interdependent interplay between E1 and E2.\n\nHCV RNA replication occurs in altered, endoplasmic reticulum (ER)-derived membrane structures known as the “membranous web” (MW). Such structures are known to be critical for RNA replication and have been observed both in vitro and ex vivo31–34.\n\nThe MW is a complex network of altered membrane structures, formed through the concerted action of several non-structural proteins34. Several lines of evidence suggest that double membrane vesicles (DMVs), which represent the major components of the MW, could represent the sites of viral RNA replication in infected cells. Indeed, the viral proteins NS3 and NS5A and the active viral replicase have been found in DMVs, along with vesicle-associated membrane protein-associated protein A and cholesterol, host factors critical for viral RNA replication34,35.\n\nDuring replication, the RNA-dependent RNA polymerase NS5B ensures the production of newly synthetized positive-strand RNA31,36,37 following generation of negative-strand RNA. Recently, 12 different structures of NS5B were crystallized during primed initiation or elongation of RNA synthesis, thus providing a unique look at the structural basis of HCV RNA replication and the inhibitory mechanism of nucleot(s)ide-analog inhibitors38.\n\nRegulation of hepatitis C virus replication by miR-122. One of the unique features of HCV replication is its requirement for the liver-specific microRNA miR-122, which enhances translation and replication through binding to the 5′ non-coding region (NCR) of HCV RNA39,40. Beyond contributing to the restricted tissue tropism of HCV for the liver, miR-122, in concert with Ago2, stabilizes the viral RNA41 and prevents its decay by the exoribonuclease Xrn142. Recent research suggests a role for another exoribonuclease, Xrn2, in miR-122-mediated prevention of viral RNA decay for certain HCV genotypes43. However, Xrn2 restriction of HCV RNA replication is likely a marginal, indirect effect observed with only a few genotypes44.\n\nRecently, several reports brought novel insights to the roles and impacts of miR-122 sequestration by HCV on viral replication and liver homeostasis. By reducing the amount of viral genomes engaged in translation, miR-122 was recently shown to increase the fraction of viral RNAs available for replication, thus enhancing RNA replication and protein synthesis45.\n\nAnother study recently reported that through the sequestration of miR-122, HCV RNA induces a global de-repression of miR-122 targets over the human transcriptome46. The authors suggested that the miR-122 “sponge effect” by HCV RNA may contribute to unbalance liver homeostasis, hence favoring the development of liver cancers.\n\nLipid peroxidation as repressor of hepatitis C virus replication. Unlike other viruses, HCV is sensitive to oxidative membrane damage, which usually occurs in stressed tissues. Lipid peroxidation affects the conformation of NS3-4A protease and NS5B, restricting HCV replication in cell culture and thus facilitating the long-term persistence of the virus within infected tissues47.\n\nThe inability of non-adapted HCV strains or patient isolates to replicate in cell culture has strongly impacted HCV research over the last decades, thus limiting cell culture assays and interpretation to a single, non-adapted molecular clone, JFH-1. More importantly, the molecular mechanisms restricting replication of non-adapted strains in cell culture were unknown. Recently, a genome-wide gain-of-function screen found that the cytosolic lipid-binding protein SEC14L2 is an HCV host factor that allows detectable replication of diverse, non-cell culture-adapted HCV replicons and molecular clones in hepatoma cell lines which do not endogenously express SEC14L248. Interestingly, the effect of SEC14L2 on viral replication was indirect, caused by an enhancement of vitamin E-mediated inhibition of lipid peroxidation. The discovery of SEC14L2 opens new avenues for the generation of non-adapted HCV cell culture systems, which could shed light on previously unknown aspects of HCV biology and genetic diversity.\n\nNovel insights into hepatitis C virus assembly. HCV particle assembly is a complex molecular process involving the recruitment of structural proteins and viral RNA at the assembly site, formation of the nucleocapsid, and the envelopment and maturation of the viral particle49. As this process involves a considerable number of viral factors, host proteins, and lipid components, the molecular mechanisms and factors regulating this process are still not fully characterized, limiting the design of DAAs targeting the late steps of the HCV life cycle.\n\np7 is a small, hydrophobic viral protein that associates as multimeric complexes to form ion channels essential for viral assembly and release50. Nevertheless, the precise molecular mechanism by which p7 regulates assembly still needs to be characterized. The recent structural resolution of the p7 ion channel has brought new insights into the potential mechanism of the action of p751. p7 displays an unusual funnel-like architecture as well as a mechanism of cation selection mediated by two pairs of amino acids. Overall, this structure provides a clearer basis for p7-mediated cation conductance and insights on developing channel activity inhibition strategies.\n\nRecently, another interesting report demonstrated that HCV’s hijacking of host innate immune signaling pathways enhances viral assembly. Indeed, indirect activation of the IκB kinase-α (IKK-α), a component of the NF-κB signaling cascade, by the 3′ NCR of HCV can activate a transcriptional program, leading to the induction of lipogenic genes and increased formation of core-associated lipid droplets52. This provides a strong link between HCV-induced innate immune responses, lipid metabolism, and disturbance of liver lipid metabolism.\n\n\nB. Hepatitis C virus humanized mice models: moving forward\n\nOwing to the narrow host tropism of HCV, restricted to humans and chimpanzees, HCV-host interactions in vivo, HCV-induced pathogenesis, and the development of anti-viral strategies have all been hindered by the lack of a tractable, cost-effective animal model for HCV infection53. As murine hepatocytes do not support HCV entry and replication, human liver chimeric mice have been a prominent model to study HCV infection in vivo over the past few decades54. However, this model is limited by donor-to-donor variability, high costs, and the immunodeficient background of the recipient mice. It was previously reported that CD81 and OCLN represent the minimal set of human factors required for HCV uptake into mouse cells in vitro17. Recently, two successive studies reported the first genetically humanized mouse model through transient expression of human CD81 and OCLN or through stable expression of the four canonical HCV entry factors (i.e. CD81, SCARB1, CLDN1, and OCLN)55,56. Blunting of anti-viral signaling allowed low-level viral replication, de novo particle production, and completion of the viral life cycle in vivo55. In the future, such a model, as well as its future refinements, will be of considerable use to further dissect HCV infection in vivo and evaluate vaccine strategies.\n\nFurthermore, an immunodeficient mouse co-engrafted with human hepatocytes and human hematopoietic stem cells was found to support HCV infection and develop T-cell-specific responses57. However, no viremia or B-cell responses could be observed, highlighting the improvements needed in this system. In the future, co-engrafted humanized mice able to mount improved immune responses will represent a unique platform for characterizing HCV infection in vivo, understanding critical immunological events regulating outcome of infection, and evaluating vaccine candidates.\n\n\nC. Immunity and pathogenesis\n\nAcute HCV infection is spontaneously cured in 20% to 30% of patients, whereas the great majority of them are unable to clear the virus and will develop a chronic infection in the face of an ongoing innate and adaptive immune response. However, the early immunological events regulating the outcome of infection are still poorly described.\n\nInnate immune responses to hepatitis C virus and viral countermeasures. The innate immune system represents the first line of defense of host cells against viral infections58. The innate immune system detects viral infection largely through germline-encoded pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs), the retinoic acid-inducible gene I-like receptors (RLRs), the nucleotide oligomerization domain-like receptors (NLRs), and cytosolic DNA sensors. Activation of these PRRs leads to the secretion of interferons (IFNs), key cytokines responsible for the establishment of an anti-viral state in cells59.\n\nDuring HCV infection, TLR3 recognizes HCV double-stranded RNA intermediates, thus inducing the production of inflammatory cytokines60. In parallel, RIG-I recognizes the poly-U/UC tract of the HCV 3′ untranslated region (UTR), hence inducing the production of IFNs61. However, recent evidence increasingly suggests that MDA-5 is also an important inducer of IFN production in HCV-infected cells62–64. Single-nucleotide polymorphisms of MDA-5 have been found to strongly correlate with the resolution of HCV infection, arguing a role for MDA-5 in the natural course of HCV infection65.\n\nIt was conventionally thought that the plasmacytoid dendritic cells (pDCs), the main IFN producers of the immune system, were able to produce IFNs by recognizing infectious particles66. However, a recent report showed that viral RNA-containing exosomes secreted by HCV-infected cells are the major immuno-stimulatory inducer of IFN secretion by pDCs via a TLR7-dependent mechanism67. Exosomes also now appear to be an HCV propagation carrier, as it has been shown that exosome-associated viral RNA can induce a productive infectious cycle in non-infected hepatocytes68,69. However, in contrast to infection with viral particles, exosome-mediated infection is presumably less effective and thus raises the question of its biological significance in vivo70.\n\nTo overcome the host innate immune system, the HCV NS3-4A protease can cleave the host adaptor proteins mitochondrial anti-viral-signaling protein (MAVS) and TIR-domain-containing adapter-inducing IFN-β (TRIF), thus inhibiting the RIG-I like receptors and TLRs-mediated type I and type III IFN signaling pathways within infected cells71–73. Recently, HCV NS4B was reported to block IFN production by disrupting STING interaction with MAVS and TBK174,75. These findings shed new light on the molecular mechanisms underlying the persistence of HCV infection.\n\nThe control of hepatitis C virus infection by interferon λ. In HCV-infected patients, genetic variations in the IFNλ locus are associated with spontaneous viral clearance and type I IFN-based treatment success76–79. However, the molecular mechanisms underlying the close association between IFNλ polymorphisms and the clinical outcome of HCV infection remain poorly characterized.\n\nA study recently highlighted an unsuspected molecular mechanism associating IFNλ3 polymorphism with HCV repression of the innate anti-viral response. An IFNλ3 mRNA carrying an unfavorable polymorphism was highly susceptible to AU-rich element-mediated decay and to binding of HCV-induced microRNAs, hence favoring repression of this IFNλ3 polymorphism80. Overall, this study provides a potential explanation of why particular IFNλ3 alleles are better regulators of HCV infection.\n\nIn another study, laser capture microdissection was used to isolate HCV-infected primary human hepatocytes displaying different IFNλ genotypes. Interestingly, hepatocytes from donors with clinically less favorable IFNλ genotypes were more permissive to HCV infection and exhibited reduced anti-viral responses compared with cells from donors with favorable alleles81. Hence, this represents additional, strong evidence that IFNλ alleles can predict the HCV permissiveness and innate immune responses of a particular host genetic background.\n\nOverall, all these findings highlight the importance of host genetic factors and of inducers of the innate immune response in determining the early events of infection. In the future, the combination of high-throughput transcriptomic and single-cell technologies with relevant in vivo experimental models could help to better characterize the molecular and immunological factors regulating the early events of infection and, ultimately, outcome of HCV infection.\n\nFailure of the innate immune system to control early events of infection induces the development of an adaptive immune response against HCV, highlighted by the generation of an HCV-specific T-cell response and the production of HCV neutralizing antibodies82. However, the immune mechanisms underlying the failure of the cytotoxic and humoral responses in resorbing viral infection and leading to a state of chronic infection are not well understood.\n\nRecently, several reports shed light on the impacts of T-cell dysfunction during HCV infection as well as on the molecular mechanisms contributing to such dysfunctions, which can favor the inhibition of long-term adaptive immune responses and thus the maintenance of chronic infection.\n\nDuring chronic infection, continuous antigenic stimulation can enhance the expression of inhibitory receptors on cytotoxic T-cells (CTLs), leading to impaired CTL functions. A recent report supported this argument in identifying a novel inhibitory receptor, prostaglandin E2, overexpressed on the CTL surface during lymphocytic choriomeningitis virus infection83. Blocking of prostaglandin E2 and programmed cell death 1 signaling improved CTL responses and favored better immune control of chronic viral infection. This evidence strongly suggests that similar mechanisms are at play during HCV infection and likely contribute to sustain chronic infection. Another study also recently highlighted how T-cell function may be impaired during HCV infection. HCV E2 protein and a short E2-coding RNA fragment were found to inhibit distal and proximal T-cell receptor-mediated signaling, respectively84. By affecting T-cell activation, HCV E2 protein and RNA may contribute to a global state of T-cell dysfunction and impaired adaptive immune responses favoring chronic infection. Consistently, CD8+ T-cell responses have been shown to be restored in patients following DAA-based, IFN-free therapy. The suppression of viral replication could disrupt the global state of T-cell dysfunction and reinstate T-cell function, which might be critical for the success of the therapy85.\n\nInteresting findings have also been reported regarding the impact of maternal immune tolerance during pregnancy on HCV-specific T-cell functions. Impaired T-cell responses induced by pregnancy have been shown to limit T-cell-mediated selective pressure on HLA-I epitope, hence stimulating the loss of escape mutations and the emergence of fitter virus86. Indeed, T-cell selective pressure was shown to be restored after childbirth along with the predominance of escape mutations. This suggests that maternal immune tolerance allows viruses with enhanced fitness to be vertically transmitted into a new host.\n\nT-cell function can also be seriously impaired by extensive regulatory T (Treg) cell expansion during HCV infection. Indeed, a recent report analyzed the HCV-specific T-cell response following HCV challenge of non-human primates previously infected with a subinfectious dose of HCV. Although subinfection induced the development of an HCV-specific T-cell response, subsequent challenge led to expansion of Treg cells that suppressed an effective T-cell and recall response87. Hence, individuals who were repeatedly exposed to HCV may be more prone to develop chronicity through exposure-induced immune suppression and strong Treg cell expansion.\n\n\nD. Curing and preventing hepatitis C: where are we going?\n\nOver the last 3 years, a combination of DAAs involving NS3-4A inhibitors, NS5A inhibitors, and NS5B nucleos(t)ide or non-nucleos(t)ide inhibitors has demonstrated their strong potency to induce a sustainable virologic response close to 90% to 100% against the most prevalent HCV genotypes2,88, thus allowing HCV patients to be cured. However, several challenges will likely appear in the future, as most of these drugs have a low barrier to resistance, with the exception of NS5B nucleos(t)ide inhibitors. Indeed, resistance-associated mutations to several DAAs have already been characterized in NS3-4A, NS5A, and NS5B as well as natural polymorphisms observed in certain genotypes and subtypes3.\n\nHowever, strategies are currently being developed to increase the resistance barrier of DAAs. Daclatasvir (DCV) interferes with NS5A functions and is a very potent HCV inhibitor, but mutations within NS5A can arise fairly easily, rendering HCV resistant to DCV’s anti-viral activity89. However, recent findings demonstrated that this resistance is overcome when DCV is used in combination with an NS5A inhibitor analogue, commonly inactive against both wild-type and resistance NS5A variants. This synergistic effect underscores the importance of inter-protein communication between NS5A molecules in the mechanisms of action of DCV. Moreover, this finding emphasizes that progress in understanding viral protein functions is critical to enhance resistance barriers of DAAs and to develop rational DAA combination therapy for effective clinical treatment90.\n\nMoreover, another critical challenge lies in the fact that curing HCV patients does not mean they are cured of liver disease or protected against the development of potential HCC. However, our understanding of the HCV-induced mechanisms leading to liver disease and cancer remains somewhat limited. Hence, a strong emphasis on these mechanisms will be required in the upcoming years to develop original therapies preventing the development of such diseases and face the need of protecting HCV-cured patients against them.\n\nThe appearance of HCC in chronically infected patients is likely to be stimulated by the immune tolerance induced by continuous antigen stimulation and T-cell dysfunction. Hence, the prevention of HCV-induced HCC requires an improved understanding of the immunological events favoring both the maintenance of HCV chronic infection and the appearance of HCV-induced liver cancers. A better profiling of the dysfunctions of the adaptive immune responses during HCV infection could provide innovative immunotherapy strategies that could reduce the risk of HCC in chronically infected patients or HCV-cured patients. Moreover, the recent success of immunotherapies against lung and melanoma cancers91 also highlights the need for improved knowledge about liver cancer immune evasion mechanisms, which could open avenues for treating HCV-free patients who develop HCV-induced HCC.\n\nDespite the clinical efficacy of the DAAs, the great majority of HCV-infected patients worldwide do not have access to these treatments because of their high cost. One solution lies in the development and production of novel, cost-effective DAAs or antibodies that could cure HCV patients. This possibility is supported by data from recent studies that demonstrated that passively administered anti-HCV envelope92 and anti-CLDN1 antibodies can cure human liver chimeric mice of chronic HCV infection93.\n\nIn parallel, a cost-effective, prophylactic, and therapeutic HCV vaccine is still urgently needed to significantly impact the number of HCV cases worldwide. However, such research has been extremely challenging over the past two decades. The incomplete knowledge of the adaptive immune response to HCV and exposure-induced immune suppression limits the design of T-cell-based vaccines. Additionally, the high E1E2 genetic variability has hampered the generation of broadly potent immunogens. Several past and recent reports have found that the generation and use of broadly neutralizing antibodies (bNAbs) targeting E1E2 can lead to efficient, pan-genotypic neutralization of HCV in vitro94,95 and even HCV clearance in vivo92. However, such bNAbs are usually poorly induced in chronically infected patients. Hence, the generation of immunogens able to trigger an effective bNAb response in patients has emerged as a considerable barrier to the generation of a potent HCV vaccine. An important obstacle in the development of such immunogens likely lies in our poor understanding of the structural and functional basis of HCV neutralization, E1E2 conformational plasticity, and epitope accessibility. The recent structural resolution of an E2core structure25,26 represents a considerable advancement for elucidating the mechanisms of HCV neutralization. However, on the surface of viral particles, it is likely that E2 takes on other conformational states, as E2core is only a partial structure and E2 conformation is dependent on its association with E1. In the future, novel insights into E1E2 structures and conformational changes will provide important avenues for the generation of an effective vaccine.", "appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nWork in the laboratory is supported in part by grants from the National Institutes of Health (1R21AI117213, 2R01 AI079031, 1 R01 AI107301, 1 R56 AI106005) and a Research Scholar Award (to AP) from the American Cancer Society (RSG-15-048-01-MPC). QD is a recipient of a postdoctoral fellowship from the New Jersey Commission for Cancer Research (DHFS16PPC007). We apologize to all colleagues whose work could not be cited because of space constraints.\n\n\nAcknowledgements\n\nWe would like to thank Jenna Gaska for her helpful discussion and comments on drafts of this article.\n\n\nReferences\n\nChoo QL, Kuo G, Weiner AJ, et al.: Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science. 1989; 244(4902): 359–62. PubMed Abstract \| Publisher Full Text\n\nGutierrez JA, Lawitz EJ, Poordad F: Interferon-free, direct-acting antiviral therapy for chronic hepatitis C. J Viral Hepat. 2015; 22(11): 861–70. PubMed Abstract \| Publisher Full Text\n\nPoveda E, Wyles DL, Mena A, et al.: Update on hepatitis C virus resistance to direct-acting antiviral agents. Antiviral Res. 2014; 108: 181–91. PubMed Abstract \| Publisher Full Text\n\nHilgenfeldt EG, Schlachterman A, Firpi RJ: Hepatitis C: Treatment of difficult to treat patients. World J Hepatol. 2015; 7(15): 1953–63. 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Proc Natl Acad Sci U S A. 2005; 102(8): 2992–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDing Q, Huang B, Lu J, et al.: Hepatitis C virus NS3/4A protease blocks IL-28 production. Eur J Immunol. 2012; 42(9): 2374–82. PubMed Abstract \| Publisher Full Text\n\nDing Q, Cao X, Lu J, et al.: Hepatitis C virus NS4B blocks the interaction of STING and TBK1 to evade host innate immunity. J Hepatol. 2013; 59(1): 52–8. PubMed Abstract \| Publisher Full Text\n\nNitta S, Sakamoto N, Nakagawa M, et al.: Hepatitis C virus NS4B protein targets STING and abrogates RIG-I-mediated type I interferon-dependent innate immunity. Hepatology. 2013; 57(1): 46–58. PubMed Abstract \| Publisher Full Text\n\nThomas DL, Thio CL, Martin MP, et al.: Genetic variation in IL28B and spontaneous clearance of hepatitis C virus. Nature. 2009; 461(7265): 798–801. 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PubMed Abstract \| Publisher Full Text\n\nFridell RA, Qiu D, Wang C, et al.: Resistance analysis of the hepatitis C virus NS5A inhibitor BMS-790052 in an in vitro replicon system. Antimicrob Agents Chemother. 2010; 54(9): 3641–50. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSun JH, O'Boyle DR 2nd, Fridell RA, et al.: Resensitizing daclatasvir-resistant hepatitis C variants by allosteric modulation of NS5A. Nature. 2015; 527(7577): 245–8. PubMed Abstract \| Publisher Full Text\n\nJiang T, Zhou C: The past, present and future of immunotherapy against tumor. Transl Lung Cancer Res. 2015; 4(3): 253–64. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nde Jong YP, Dorner M, Mommersteeg MC, et al.: Broadly neutralizing antibodies abrogate established hepatitis C virus infection. Sci Transl Med. 2014; 6(254): 254ra129. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMailly L, Xiao F, Lupberger J, et al.: Clearance of persistent hepatitis C virus infection in humanized mice using a claudin-1-targeting monoclonal antibody. Nat Biotechnol. 2015; 33(5): 549–54. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGiang E, Dorner M, Prentoe JC, et al.: Human broadly neutralizing antibodies to the envelope glycoprotein complex of hepatitis C virus. Proc Natl Acad Sci U S A. 2012; 109(16): 6205–10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLaw M, Maruyama T, Lewis J, et al.: Broadly neutralizing antibodies protect against hepatitis C virus quasispecies challenge. Nat Med. 2008; 14(1): 25–7. PubMed Abstract \| Publisher Full Text" }	[ { "id": "12258", "date": "04 Feb 2016", "name": "Glenn 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", "responses": [] }, { "id": "12259", "date": "04 Feb 2016", "name": "Volker Lohmann", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-131
https://f1000research.com/articles/5-130/v1	03 Feb 16	{ "type": "Opinion Article", "title": "Towards an open science publishing platform", "authors": [ "Vitek Tracz", "Rebecca Lawrence" ], "abstract": "The way science and research is done is rapidly becoming more open and collaborative. The traditional way of publishing new findings in journals is becoming increasingly outdated and no longer serves the needs of much of science. Whilst preprints can bring significant benefits of removing delay and selection, they do not go far enough if simply implemented alongside the existing journal system. We propose that we need a new approach, an Open Science Platform, that takes the benefits of preprints but adds formal, invited, and transparent post-publication peer review. This bypasses the problems of the current journal system and, in doing so, moves the evaluation of research and researchers away from the journal-based Impact Factor and towards a fairer system of article-based qualitative and quantitative indicators. In the long term, it should be irrelevant where a researcher publishes their findings. What is important is that research is shared and made available without delay within a framework that encourages quality standards and requires all players in the research community to work as collaborators.", "keywords": [ "open science platform", "preprint", "impact factor", "open access", "open data", "life sciences", "publishing" ], "content": "Introduction\n\nThe way science and research is done is evolving rapidly. The change is characterised by more open, collaborative and networked ways of sharing information and making discoveries. This change is being driven by recognition of the profound benefits to the pace of scientific progress that can be brought by collaboration and ready exchange of ideas between and beyond disciplines and sectors. Furthermore, technology can now enable and support collaboration, information sharing and rapid data exchange and analysis. To make science more efficient, we need to remove the waste in the current system, as exemplified by ongoing debates concerning research duplication and the growing doubts about the reproducibility of findings.\n\nThe goal for open science is to accelerate scientific progress and to turn what is discovered into benefits for all. An essential part of this is to ensure that scientific findings are open and available for scrutiny, rapidly accessible, and easily discoverable for others to use and build upon. The way research findings are currently made available – through journals – is increasingly at odds with the aspirations of open science.\n\n\nJournals: an outdated mechanism for publishing work\n\nThere is some recognition amongst the research community that journals are now an outdated method for publishing new research findings and no longer serve the needs of much science1,2. The current publishing processes bring many problems that are not conducive to the progress of science. These include:\n\n• Holding up science. The selection process that journals run to help them decide what to accept is typically done before publication, and hence leads to a delay in the availability of new findings to those who need them3. There are no obvious benefits of such a delay, and indeed it can sometimes cause significant damage when the health of patients are affected4.\n\n• Non-transparent. The peer review scheme used by most journals is anonymous (the choice of referees is hidden from the authors, and the referees’ comments are not always fully shared with the authors). Its current main function is usually to help editors make decisions on what to publish and what to reject, rather than to help the authors improve their article. Furthermore, the readers do not get the benefit of the insight on any outstanding issues the referees may have identified with the article along the way. In a competitive research environment, these non-transparent schemes can lead to abuse of the system in a variety of different ways5,6 and there is little to stop this from happening.\n\n• Much science never shared. Many findings are currently not published (such as small studies, data and software papers, negative and null studies etc), which often leads to significant research waste and potential publication bias7–9. This is often caused by the fact that journals, in order to maintain their Impact Factor, are keen to attract submissions that bring in more citations10.\n\n• Waste in the system. There is significant waste in the publishing system caused by articles moving from journal to journal until they find somewhere that will accept to publish the article. This brings inefficiencies in the system and wasted effort both for the authors and for the referees in repeated refereeing.\n\n• Too expensive. Most new scientific findings are still published in subscription journals that are usually expensive. This means that a large proportion of the community (researchers and the public) cannot access the research11,12. Open access journals certainly enable everyone to have access but their Article Processing Charges are still often very high13. So-called hybrid journals (subscription journals that require authors to pay for an open access option) are even worse, because they create extra costs for the information exchange system and significantly slow the growth of fully open access journals.\n\nWhich journal an article is published in is still commonly used as a surrogate quality measure for an individual article and its authors. However, it is well established that such use, specifically of a journal’s Impact Factor (JIF), is an inappropriate and misleading indicator of either the importance and/or quality of a specific article14–17, or of the potential of the author(s) as researchers18.\n\nThe reality is that journals are not essential anymore (though sometimes useful) for the discovery of research results. Much more efficient tools and services can and are being developed using the information in citation databases such as PubMed (for biomedical research) and/or Google Scholar, Scopus, Web of Science etc., to help researchers find new articles in an area of interest. Journals survive primarily because they are needed by authors to get the reflected benefit of the JIF. One of the challenges for the future is to develop reliable and effective qualitative assessment of both research articles and an individual researcher’s scientific output.\n\nA new way of publishing, discussing and reviewing new scientific findings is urgently needed to speed up the progression of science, and to improve the fairness of the system used to judge researchers with regards to their next grant or career move. Such a new system should also enable funders to maximise the value of their research investment. The technology to enable such a change is now available. This new approach needs to avoid the significant delays in making new findings visible, and needs to be efficient, easy to use and not expensive.\n\nIn our view, and as we outline further below, such a new process would only work if driven by the authors within a scientific framework that facilitates self-regulation. There needs to be a generally agreed set of ethical and technical rules, and these should be overseen by bodies directly representing researchers, such as funders, institutions, organisations and societies.\n\n\nPreprints: the benefits and limitations\n\nOne idea currently being discussed is the much wider use of preprints in the life sciences, i.e. the online posting of an article to make it openly visible and citable prior to peer review19–21. One of the features of preprints is that authors remain completely in control and no formal refereeing is required. The culture of using preprint servers in physics (arXiv) has established itself well without structured refereeing. It is surprising that preprints and journals have survived alongside each other for so long, and that authors still feel the need to have the preprint converted into an article published in a journal, even though all who need to have already accessed the article from the preprint server. It seems that even here, where the article is published can have a significant impact on the prospects of the authors’ career. This perhaps illustrates the powerful hold that journals and JIFs have on researchers’ careers. Preprints, even when used widely, may not remove this dependence.\n\nDespite a modest increase in the use of preprints in life sciences recently with the launch of PeerJ PrePrints and BioRxiv, the overall use of preprints to-date in the life sciences is still not in significantly large numbers (under 5,000 preprints posted on these two sites to-date, compared with over 1.13 million articles added to PubMed just in the past year). There have been several attempts to encourage the use of preprint servers, but none has really taken off. In the early days of open access back in the late 1990s, the idea had been to start with a preprint server, but there was such strong objection from some members of the PubMed Central (PMC) National Advisory Committee in its first meeting22, that a decision was taken not to accept preprints. The prevailing culture within the research community at the time was not to recognise preprints as a formal output, and it is not clear how different the culture really is now.\n\nThere are many benefits to authors and to the community of using preprint servers as part of the mix of options in publishing life science research articles. Perhaps most important is the immediacy of placing research findings on public record (also thereby establishing some level of priority). Preprints can also be used to gather informal comments from colleagues on possible problems and potential improvements before committing to the non-transparent refereeing as operated by most journals. But preprints solve only some of the problems outlined earlier, and technology now enables us to do much more with findings at the stage that researchers are willing to share them.\n\nMany questions have yet to be answered about the adoption of preprints in the life sciences. Among them:\n\n• Will a significant proportion of authors opt to post preprints?\n\n• Will enough colleagues and other researchers comment openly (or even confidentially) on articles posted there, to make the effort useful? A quick review of a randomly selected block of 100 articles posted on BioRxiv in June/July 2015 (so over 6 months ago to ensure time to receive comments) showed that there were only two preprints in that selection that had any external comments, each being back and forth with a single commenter.\n\n• Will subsequent formal submission to a journal and progress through the lengthy process of official peer review as currently operated by journals still be required, thereby retaining many of the problems outlined earlier?\n\nWhat we propose here is a scheme that takes full advantage of the benefits that preprints can bring, combined with a new type of invited, formal transparent peer review that differs significantly from the one currently run by most journals, both in its goals and in its processes. The description of this scheme (which has been running for three years now in the publication process of F1000Research) is described below.\n\n\nA new experiment: Open Science Platform\n\nHere we present a model as a starting point that we envisage will evolve as researchers embrace the opportunity to share their findings and data in new ways. Our guiding principle is that open science publishing should be author-driven to enable researchers to share openly and rapidly any new findings that they think are worth sharing. Findings should be published near immediately, in a format most appropriate to convey the information in the discovery. In addition, publication should be usually followed by post publication, formal invited peer review, that is conducted transparently. This is both to help authors to improve their presentation and to provide auditable qualitative assessment of the research.\n\nWriting and submission. The process of compiling findings, writing accompanying narrative and making this available for public view and scrutiny can be simplified by the use of new improved software. These tools can help identify relevant papers through increasingly powerful learning algorithms (e.g. F1000Workspace, Mendeley, Readcube). They can also enable collaborative authoring (e.g. F1000Workspace, Overleaf, Google docs), and provide formatting tools to simplify the process of structuring an article to ensure all the necessary underlying information has been captured (e.g. F1000Workspace, EndNote). Submission for posting as a preprint, and/or for formal publication and peer review, should be as simple as a single click.\n\nInitial objective checks. We envisage that all submitted articles should be rapidly screened against a set of objective criteria. Such criteria might include checks for obvious non-scientific content, readability, ensuring the work is not plagiarised, that it meets standard ethical requirements, and that the underlying data has been supplied together with detailed methods. They could also include other specifics as agreed by the relevant communities depending on, for example, the type of experimental study being described. The specifics of these checks should be listed transparently.\n\nFAIR (Findable, Accessible, Interoperable and Reusable) data. The need for an open data policy seems undisputable to us: the data underlying the findings presented in the article should be openly accessible, together with information on how the data were analysed such as the software used etc., so that users can fully scrutinise the presented findings and repeat the work, if they wish. The data and any code should be in a form that can be used by the referees and readers, together with detailed methods as to how it was generated. They need to be stored in approved repositories that meet a minimum set of criteria to ensure long-term availability and persistence, with appropriate levels of protection for sensitive data. How much and what data to provide is a highly complex issue and will require specific instructions to be developed by the relevant communities, together with more generalised requirements on data format, structure and associated metadata. There are numerous groups working collaboratively worldwide on these many issues such as the Data Fairport Initiative, FORCE11, Research Data Alliance and others.\n\nPublication. Any submitted article that passes these rapid checks would then be published (made public) immediately, given a unique identifier (making them permanently citable) and clearly labelled as not yet peer reviewed. Following the initial screening, we think it is important that there is no editorial decision on accepting or rejecting research articles, to remove the inherent biases in having a single Editor making a decision on behalf of the rest of the community, and to help to remove publication bias.\n\nIdentifying referees. In order to facilitate communication between peers without the interference of editors, and to ensure peer review is carried out by qualified experts, we envisage that authors should select referees from a large community of recognised experts (potentially with the assistance of algorithmic tools), as long as they abide by a clear set of transparent rules and criteria on how to select suitable referees. Both authors and referees should also transparently declare any conflicts they have with each other or the work being refereed.\n\nQuestions remain around how this community should be defined. Should a database (growing and changing) be created of ‘approved referees’? How does someone qualify to be included in this community of approved referees? How will this database grow, and who will control it? For example, it could comprise grantees of major granting bodies with some minimum experience/publication record. Or it could be constructed like a large virtual faculty like the F1000 Faculty. It would seem that there should be greater collaboration across the key stakeholders involved in research (funders, publishers, research institutions, researchers, industry) to work together to resolve these issues.\n\nThe peer review process. Referees should then be invited as requests from the authors, but mediated by the platform. Without the need to select for impact, the peer review process can refocus on its basic goal to help the authors improve their work and to provide valuable context and feedback on the viability and quality of the published research for the reader and for anyone reviewing the work of that individual.\n\nReferees should be given a set of clear instructions and guidance on what aspects of the article to assess and what is expected in a referee report (as common practice today). Referees should also be able to benefit from tools similar to those provided to authors, to make the writing of the report more efficient. With the publication of new types of findings, there is an interesting question about whether all findings need to be refereed (for example, short commentary articles). There are also many questions about what types of aspects of an article can a referee sensibly be expected to check within a reasonable timeframe, especially with regards to data, code and figures. Should peer review differ for different types of findings?\n\nGiven the open questions about what level of peer review should be required for different article types and for data and software, there are also questions around what constitutes an article being ‘peer reviewed’. PubMed has developed criteria for F1000Research and future publishing platforms23, but should this always be the same irrespective of the type of finding?\n\nWe think it is very important that all peer review reports are published transparently alongside the name of the referee – open peer review has been repeatedly shown to be of comparable (if not better) quality, and also often more constructive compared with closed peer review24,25. The authors would drive the process via the platform provider, so that they can engage in open discussion with the referees and can revise their article and publish new findings as and when they feel appropriate. This process should continue until the authors wish to stop. All versions need to be independently citable but connected, and a dynamic citation26 can be used to ensure the reader is always clear about the article version and its peer review status. In addition to formal refereeing, any researcher should be able to openly comment and discuss an article in a transparent way, although this should not impact the formal peer review status of the article.\n\nBenefits for referees. Referees currently receive very little direct benefit from the process of refereeing and their contribution is currently not visible. We believe referees should receive real benefits for contributing to what is a crucial function in improving the work of others. Referee reports should receive their own persistent identifier (digital object identifier – DOI) and therefore be independently citable which means that referees can receive their own metrics. Refereeing can now be included as a formal contribution on Publons and on ORCID profiles27 (the researcher unique persistent digital identifier), and we would urge institutions and funders to lay out an expectation (and provide formal recognition), for their grantees to contribute to this important process. Are there other forms of credit that referees could receive for their important role? Should ways be developed to qualitatively assess the work of referees?\n\nAccess and cost. All articles should be published (made visible and citable) using immediate open access so that everyone has equal access to new findings. The cost of running the process described above is considerably cheaper than the traditional process as it removes the substantial costs associated with editorial decision making. There are of course still costs involved in running the peer review process, conducting the initial set of checks, and building and maintaining the tools required to operate such a system. These cheaper costs would still be covered as now by research funders, and competition between service providers should put further downward pressure on these costs. Where there is no research funding, there needs to be further consideration across all stakeholders as to how best to cover the modest fees, whether through institutional funds or other sources.\n\n\nIndicators of quality and importance\n\nIt remains important that there are indicators of the value, importance, use and re-use of research findings and data. Research outputs, in all their forms, are valuable indicators of research and knowledge progression, as well as of the ‘performance’ and productivity of scientific fields and of the researchers who are generating those outputs. Such indicators are also vital for users of research findings, such as health professionals and policy makers, to help get relevant research findings into policy and practice more effectively and without unnecessary delay.\n\nThe indicators that are adopted to provide a view on research must be meaningful, contextualised and used responsibly28. The Leiden Manifesto29 recently emphasised the importance of combining quantitative and qualitative indicators in assuring a balanced and robust conclusion about the value of specific research. Furthermore, the selection of indicators that are used in any assessment should be tailored according to the purpose of the assessment. We should all seize the opportunity of working in a different publishing system to respond to recommendations such as those in the Manifesto, and redress our reliance on erroneous and misleading measures of research quality. Improvements in our ability to identify, track and analyse the outputs means that we can also shift our emphasis away from a reliance on ‘metrics’ based solely around the academic citation of a research paper and its hosting journal (e.g. JIF), as supported by signatories of DORA30.\n\nOpen peer review can play an important part in this, as researchers can gain visibility and credit for their contribution to the progression of another’s work. Furthermore, transparent refereeing provides researchers, and potential users of research, with another marker of quality as a peer reviewer’s credentials and what they say about a piece of research can become part of the assessment – instead of hidden and lost from the public record. A more appropriate use of citation-based indicators should also be included in measuring quality, such as the Relative Citation Ratio (RCR) recently proposed by the NIH31, remembering that citation-based measures take considerable time, which may be an issue particularly for younger researchers. Post-publication identification of interest and importance of an article, and commentary about the context and potential implications of the findings should become a key aspect of science journals in the future, and could of course play a role in this qualitative assessment of research.\n\nIt is, of course, not usually possible to predict the longer term impact or consequence of a new discovery at the time of publication and refereeing (e.g. DNA fingerprinting, monoclonal antibodies), and so it remains important that reflections on the significance of research can be done at any time in the future (as is the practice on F1000Prime, PubMed Commons etc).\n\n\nWhat next?\n\nIndividual elements of what is described above have been developed by many groups. For example, there has been a steady rise in the use of open peer review since the launch of the medical BMC-series journals in the early 2000s followed by BMJ Open, both using mandatory open peer review, and others have followed suit more recently offering opt-out open review such as Nature Communications. However, because this process is still conducted before the article is made publicly available, the peer review history is only made visible for those articles that ultimately get accepted and still hides the reasoning behind any decisions to reject articles. The increasing discontent amongst researchers and the scientific community as a whole has given rise to new approaches such as eLife, and both they and PLOS have tried to make a stance against the JIF by vowing to never advertise their JIFs – this of course does not prevent the use of journal titles in making decisions that affect a scientists’ future. There has also been a rise in the prominence of data as a key element of publication, with the launch of data journals such as GigaScience and Scientific Data, and more stringent data policies for existing journals such as the PLOS ONE’s data policy, released in 2013 and adjusted in 2014.\n\nThe elements combined together into a single platform as described above has already been developed and is in active use by thousands of scientists through F1000. This combines the open science publishing platform F1000Research (the option to initially post an article as a preprint for general community comment will be added very shortly), with tools to assist in writing in F1000Workspace, and some measures of qualitative assessment of published articles through F1000Prime, both to inform reading and to help assess new findings after publication. Many other publishers have begun to create similar platforms that would compete to provide such services to researchers and funders.\n\nHowever, a widespread change to a new way of publishing is unlikely to happen whilst the research community relies primarily on journals to provide the outlet for research findings. We therefore propose that to enable open science to succeed, researchers should be able to publish any research data and findings that they consider to be useful to others and to publish it without delay. To achieve this, we believe that there needs to be a fundamental shift in the way research findings are shared.\n\nPublishers and others can support this process by providing services that meet a community-agreed set of rules (such as those suggested above); competition between providers will naturally lead to improved services and reduced costs. Meanwhile, journals could begin to provide qualitative assessment and to encourage discussion of findings published on these platforms, for example like Nature magazine’s News & Views section or Current Biology’s Dispatches. Funders are perfectly placed to help drive this shift by approving those providers that offer a high quality service that meet the agreed requirements. In time (as with open access), we anticipate that making research findings and data available in this open science way will ultimately become the norm and a requirement of all public funders. Researchers would then be free to choose whichever of the approved services they prefer, and articles would also be available to all on mirrored repositories (as open access articles are now available on PubMed Central and Europe PubMed Central), together with all their versions, referee reports and others comments, and the supporting data.\n\nIn the long term, it should be irrelevant where researchers publish their findings. What is important is that to speed up scientific progress, discovery and impact, research should be shared and made available without delay for others to use and to build upon. Making findings available needs to be done within a framework that encourages quality standards and requires all players in the research community to work as collaborators.", "appendix": "Author contributions\n\n\n\nBoth VT and RL conceived and wrote the article, and approved the final version.\n\n\nCompeting interests\n\n\n\nVitek Tracz is the Chairman and founder of F1000, and Rebecca Lawrence is the Managing Director of F1000, which is comprised of F1000Research, F1000Workspace and F1000Prime.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nKrumholz HM: The End of Journals. Circ Cardiovasc Qual Outcomes. 2015; 8(6): 533–534. PubMed Abstract \| Publisher Full Text\n\nBishop D: Will traditional science journals disappear? 2015. Reference Source\n\nBjörk B, Solomon D: The publishing delay in scholarly peer-reviewed journals. J Informetr. 2013; 7(4): 914–923. Publisher Full Text\n\nWHO: Developing Global Norms for Sharing Data and Results during Public Health Emergencies. Reference Source\n\nOransky I: 17 retractions from SAGE journals bring total fake peer review count to 250 - Retraction Watch blog. 2015. Reference Source\n\nMoylan E: Inappropriate manipulation of peer review - BioMed Central blog. 2015. Reference Source\n\nChalmers L: Underreporting research is scientific misconduct. JAMA. 1990; 263(10): 1405–8. PubMed Abstract \| Publisher Full Text\n\nChalmers I, Dickersin K: Biased under-reporting of research reflects biased under-submission more than biased editorial rejection. [version 1; referees: 3 approved]. F1000Res. 2013; 2: 1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSenn S: Authors are also reviewers: problems in assigning cause for missing negative studies. [version 1; referees: 4 approved]. F1000Res. 2013; 2: 17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNature Publishing Group (NPG): Author Insights 2014. Figshare. 2016. Reference Source\n\nButler D: Dutch lead European push to flip journals to open access. Nature. 2016; 529(7584): 13. PubMed Abstract \| Publisher Full Text\n\nOA monitoring. Research Information Network. 2015. Reference Source\n\nThe Reckoning: An Analysis of Wellcome Trust Open Access Spend 2013–14. Wellcome Trust Blog. Reference Source\n\nCasadevall A, Fang FC: Causes for the persistence of impact factor mania. MBio. 2014; 5(2): e00064–e00014. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrembs B, Button K, Munafò M: Deep impact: unintended consequences of journal rank. Front Hum Neurosci. 2013; 7: 291. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCasadevall A, Fang FC: Impacted science: impact is not importance. MBio. 2015; 6(5): e01593–e01515. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLozano G, Larivière V, Gingras Y: The weakening relationship between the impact factor and papers’ citations in the digital age. J Am Soc Inform Sci Technol. 2012; 63(11): 2140–2145. Publisher Full Text\n\nSahel JA: Quality versus quantity: assessing individual research performance. Sci Transl Med. 2011; 3(84): 84cm13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVale RD: Accelerating scientific publication in biology. Proc Natl Acad Sci U S A. 2015; 112(44): 13439–13446. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCurry S: The power of preprint. The Biologist. 2015; 62(6): 7. Reference Source\n\nLauer MS, Krumholz HM, Topol EJ: Time for a prepublication culture in clinical research? Lancet. 2015; 386(10012): 2447–2449. PubMed Abstract \| Publisher Full Text\n\nThe Remarkable Transformation of E-Biomed into PubMed Central. Reference Source\n\nF1000Research FAQs. Reference Source\n\nvan Rooyen S, Godlee F, Evans S, et al.: Effect of open peer review on quality of reviews and on reviewers’ recommendations: a randomised trial. BMJ. 1999; 318(7175): 23–27. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKowalczuk MK, Dudbridge F, Nanda S, et al.: Retrospective analysis of the quality of reports by author-suggested and non-author-suggested reviewers in journals operating on open or single-blind peer review models. BMJ Open. 2015; 5(9): e008707. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nF1000Research - How it Works. Reference Source\n\nPaglione L, Lawrence R: Data exchange standards to support and acknowledge peer-review activity. Learn Publ. 2015; 28(4): 309–316. Publisher Full Text\n\nWilsdon J, Allen L, Belfiore E, et al.: The Metric Tide: Report of the Independent Review of the Role of Metrics in Research Assessment and Management. UK: HEFCE; 2015. Publisher Full Text\n\nHicks D, Wouters P, Waltman L, et al.: Bibliometrics: The Leiden Manifesto for research metrics. Nature. 2015; 520(7548): 429–431. PubMed Abstract \| Publisher Full Text\n\nSan Francisco Declaration on Research Assessment: Putting science into the assessment of research. 2003. Reference Source\n\nHutchins BI, Yuan X, Anderson JM, et al.: Relative Citation Ratio (RCR): A new metric that uses citation rates to measure influence at the article level. bioRxiv. 2015. Publisher Full Text" }	[ { "id": "12434", "date": "15 Feb 2016", "name": "Johanna R. McEntyre", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 outlines a new model to making scientific findings public. It argues that typical journal publishing processes are no longer fit for purpose and discusses pros and cons of preprints, before describing \"Open Science Platform\". The article is very timely given the latest discussions around preprints in biology, stimulated by Ron Vale's preprint, then article on the subject (Ref. 19 in this article). Open Science Platform is in essence a nose-to-tail set of services that enable researchers to collaboratively write, publish, openly peer review, revise, credit, evaluate and archive scientific articles with minimal friction. The philosophy is that anything we think would be useful to publish should be published, and we will organize this bulk by devising better filtering/discovery mechanisms and reward systems post publication. While quite a few journals have touched many parts of this particular elephant, there is not to my knowledge an existing public platform that offers all these steps aside from the one described here. F1000 has developed such a platform, but the authors note that many other publishers are probably also building similar platforms to provide some healthy competition on the services provided. I have no major criticisms of the article itself - it is not overly pushing a particular (F1000) product but rather painting a picture of what open science publishing might look like, reviewing challenges and possible solutions to the sub-optimal aspects of our current publishing systems. Frequent reference is made to many of the on-going efforts in this area and the authors clearly state their roles at F1000. An important issue that was given only brief attention is the matter of data and other research objects associated with articles. Open science extends to many aspects other than article publishing. The authors acknowledged that data underling assertions in articles are critical, as is the need to deposit data in community-supported repositories with data-appropriate access, but delegates this particular challenge to other groups. Clearly research communities and resources need to lead from different angles - but nevertheless in a more rapid and expansive publishing mode, new mechanisms will be required to ensure rigour, and support the desired behaviour around data deposition and archiving. One of the key points of open science is to enable reuse of data and other outputs in other contexts. Therefore an open science platform for articles will need to operate in a well-coordinated fashion with data archives and processes to ensure that the gains in article publishing also apply to related data and other research objects (and the emerging credit systems for those too). One aspect I would have liked to have seen the authors explore more is the fundamental matter of how to stimulate uptake of open science platforms by the key target audience: researchers. The technical challenges are tractable, the social ones the blocker. For many researchers, deeply engaged in their work at the bench, such open science systems are a long way from current operating practices, so adoption may take a while and many incentives along the way required.", "responses": [] }, { "id": "12629", "date": "24 Feb 2016", "name": "Gary G Borisy", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe printing press, the disruptive technology of 400 years ago, revolutionized how science was communicated. Journals, along with editors and reviewers—the peer review process--eventually replaced an author-directed process of letter writing. A new disruptive technology has placed us in the midst of another revolution in communication. The power of the internet has enabled communication with a rapidity, connectivity and interactiveness unlike anything possible with print media. It therefore behooves the research community to rethink how the science communication process might be structured. Tracz and Lawrence present to us their rethinking. They consider journals to be an outdated mechanism for publishing science and they enumerate the multiple problems familiar to researchers—the delay, the non-transparency, the tussles with reviewers and editors, the jockeying for high-impact journals. Tracz and Lawrence ask if journals are really necessary anymore. In so many other areas of 21st century life, the internet has enabled the elimination of intermediaries between the end consumer and the producer. Are we at a similar stage for publications? Tracz and Lawrence propose an Open Science Platform that enables authors to publish essentially immediately after they consider their work ready to be shared with the scientific community. The work is then subjected to a post-publication review process which is transparent because the reviewers are not anonymous and their critiques as well as the authors responses are posted as an integral part of the publication process. A key issue is how the reviewers will be selected. Tracz and Lawrence propose that the authors be empowered to select and invite reviewers from a pool of qualified researchers. Of course, this shifts the question to how the reviewers become qualified and who makes this decision. The authors suggest that a virtual faculty such as that of F1000 could serve this function. Researchers would be incentivized to deliver thoughtful reviews not only because they would be identified but because their reviews would be citable documents in their own right. This would provide a benefit to reviewers which is lacking in the current system.\n\nA question that will spring to the lips of every researcher is how, in such a system, will we find the important publications. Tracz and Lawrence say that, in the long run, it should be irrelevant where authors publish their findings. But there are so many publications and so little time. Don’t journals with their editorial boards serve as key gatekeepers? Doesn’t the much-maligned impact factor nevertheless serve as a helpful guide? The Open Access movement has greatly facilitated the dissemination of research results but it has had the unintended consequence of also facilitating the emergence of predatory journals whose business model is to provide publication for pay with only the appearance, not the reality, of bona fide editorial quality control. This only compounds the problem. Indicators of the importance of publications will remain necessary. The Open Science Platform does not, per se, provide the indicators but it does provide a framework in which quantitative and qualitative metrics of importance such as citations, downloads, blogs and commentary can be incorporated into the platform. In principle, these article-based metrics can be made discoverable and substitute for the classic journal gatekeepers. Tracz and Lawrence have done the research community a service by proposing this bold, new Open Science Platform. Apprehensions aside, we have crossed into the 21st century and there is no turning back.", "responses": [] } ]	1	https://f1000research.com/articles/5-130
https://f1000research.com/articles/5-128/v1	02 Feb 16	{ "type": "Case Report", "title": "Case Report: Myelodysplastic syndrome- associated myeloid sarcoma: an unusual clinical presentation of a rare disease", "authors": [ "Emoke Horvath", "Smaranda Demian", "Elod Nagy", "Emoke Horvath", "Smaranda Demian" ], "abstract": "Myeloid sarcoma results from the extramedullary homing and proliferation of immature myeloid precursors. We present the timeline, events and diagnostic pitfalls related to a 66 year-old male patient’s case, admitted to the Hematology Clinic for pancytopenia, fever, weight loss and fatigue. The severe cytopenia and the few blasts observed in his blood smear indicated a bone marrow biopsy. The bone marrow showed hypercellularity and multilineage dysplasia with the presence of 15% myeloblasts. After the biopsy, he promptly developed paraplegia and nuclear magnetic resonance revealed an epidural tumour which was then resected.In the epidural tumour mass blast-like, round cells were observed with a complex immunophenotype, characterized by myeloperoxidase, CD117, CD15, CD99, leucocyte common antigen positivity and a high Ki-67 proliferation index. Considering the main differential diagnostic issues, the final diagnosis was stated as myelodysplastic syndrome-associated myeloid sarcoma. The prognosis was unfavourable, the bone marrow was quickly invaded by proliferating blast cells, and despite chemotherapy attempts, the patient died.", "keywords": [ "myeloid sarcoma", "myelodysplastic syndrome", "myeloid blasts", "immunophenotype" ], "content": "Introduction\n\nMyeloid sarcoma is a rare malignant solid tumour resulting from the extramedullary proliferation of myeloblasts or immature myeloid cells, that usually precedes or accompanies acute myeloid leukaemia (AML), chronic myeloproliferative disorders, sometimes presenting as the first sign of AML1. Every anatomical site of the body can be involved, but most common are the soft tissues of the head and neck, bone, skin and less often the genitourinary tract, the central nervous system and the spinal cord2,3. We present a case with a complex clinical manifestation resulting from the unusual localization of the tumour mass, and that coincides with the MDS involving simultaneously the bone marrow tissue.\n\n\nCase presentation\n\nAn otherwise healthy, 66-year-old man was admitted to the Haematology Clinic of the Emergency County Hospital (Targu-Mures, Romania) in a poor general condition, presenting fatigue, continuous fever, night sweating and weight loss (10 kg in 3 months), with symptoms occurring for a few weeks. His past medical history was unremarkable, without a known history of toxic or drug exposure. Physical examination evidenced a mucocutaneous pallor, but no lymphadenopathy or organomegaly. Acute myeloid leukaemia/myelodysplastic syndrome was suspected. Baseline laboratory data confirmed symptoms of ineffective haematopoiesis. Peripheral blood examination revealed pancytopenia: severe anaemia, trombocytopenia and leukopenia with 2% blasts of myeloid lineage (Table 1), associated with elevated serum LDH (1250 U/L, reference range 240–480 U/L). The low-level presence of blasts in the blood smear (2%) indicated a bone marrow biopsy in order to establish the diagnosis, which was effected on the second day of hospitalization. Post-biopsy, after a few hours he developed paraplegia. Cerebrospinal nuclear magnetic resonance was recommended and performed, which elucidated an epidural tumour mass of the thoracic spine (suspected metastasis), compressing the dural sac in the spinal channel at the T1–T3 levels. No other tumoral formation was found by complex imagistic investigation. On the same day, the patient underwent an emergent laminectomy, the tumour being totally resected. The bone marrow biopsy and the tumour fragments were submitted for histopathological examination.\n\nThe hypercellular bone marrow biopsy showed a multilineage dysplasia characterized by dyserythropoiesis, dysgranulopoiesis and dysmegakaryopoiesis with approximately 15% CD34/CD117 positive myeloblasts without Auer rods, associated with the presence of abnormal localisation of immature precursors (ALIP). Based on this histologic picture, according to the clinical and laboratory data, our diagnosis was refractory anaemia with excess blasts-2 (RAEB-2). The spinal tumour fragments showed a diffuse proliferation of undifferentiated myeloblast-like round tumour cells, mixed with a few neutrophil precursors.\n\nConsidering the two histological findings, myeloid sarcoma was suspected, but for a differential diagnosis lymphoblastic lymphoma, Burkitt lymphoma, diffuse large B-cell lymphoma and small round cell tumours also had to be excluded. A large immunohistochemistry panel was helpful in achieving differentiation of these conditions. The monomorphic tumour cells revealed positive stainings for Leucocyte Common Antigen (LCA), myeloperoxidase (MPO), CD15 and CD117, associated with a high Ki67 index (over 60%). Neuroendocrine and lymphoid markers, pan CK and also CD68 were negative. In contrast, a significant number of tumour cells expressed CD99 antigen.\n\nThe histopathological diagnosis has been stated on the basis of the two biopsies, being completed to “Myelodysplastic syndrome (RAEB-2) associated myeloid sarcoma” of the spinal cord.\n\nThe clinical course was unfavourable, at the end of the first week post-surgery, the peripheral blood and bone marrow suddenly showing a burst of the blast count up to 50%. Chemotherapy with high-dose cytosine arabinoside proved to be inefficient, and the patient died at 5 weeks from admission.\n\n(a–f). Hypercellular bone marrow biopsy with reduced fatty cells, hematoxylin and eosin staining, magnification 4X (a), in the central portion of the bone marrow with foci of ALIP and dysplastic features in granulocytic lineage, hematoxylin and eosin staining, magnification 20X (b). Spinal tumor mass with diffuse and infiltrative population of large malignant cells (myeloblasts), with abundant cytoplasm and large nuclei, hematoxylin and eosin staining, magnification 10X (c), many of them with CD99, magnification 20x (d), and CD45 antigen expression, magnification 10X (e). Tumour cells showing strong MPO positivity, magnification 20X, 2', 3-diaminobenzidin (DAB) immunostaining (f).\n\n\nDiscussion\n\nThe exact prognosis of myeloid sarcoma is difficult to determine, but in general is poor1. The first description of “myeloid sarcoma” appeared in literature in 1811, and in 1904 its association with acute leukaemia was identified4.\n\nThe term \"granulocytic sarcoma” was introduced in 1967 by Rappaport to describe only tumours of granulocytic origin, however the term is now often applied to any tumour related to acute leukaemia or myelodysplastic syndrome (MDS)5.\n\nThe mechanisms of extramedullary involvement are not fully understood, but it was demonstrated that leukaemic cell surface markers (CD56) are implicated in extramedullary homing5.\n\nThe particularity of our case consists of the unusual tumour localization with spinal compression and neurologic signs, and the combination with the deficient haematopoiesis, that raises the suspicion of a metastasis in the bone marrow. Regarding phenotype, the positivity for CD45 and CD99 with a high Ki-67 proliferation index could suggest a lymphoma, but the morphological similarities between the extramedullary tumour cells and the bone marrow blasts are suggestive for myeloid sarcoma. For an accurate approach, a large-scale immunohistochemistry panel, containing mandatorily the CD45, MPO, CD15, CD79a, CD3, CD34, CD117, CD99, TdT and Ki-67 proved to be useful. The major diagnostic pitfalls are represented by distinction from lymphoblastic lymphomas (TdT+/CD34+/CD99+/CD45+/high Ki-67 index), and small round-cell tumours (CD99+/TdT-). Seifert et al. analyzed 12 cases of myeloid sarcoma and found a consistent positivity for CD117, CD43, MPO, CD68, CD34 and a sporadic reaction of leucocyte common antigen6. Two major issues of the correct diagnostic orientation are the recognition of a possible non-lymphoid CD45 positivity and ruling-out of the aberrant CD99 antigen from the pivotal markers in this tumour type. On the other hand, MPO positivity is a prominent feature for a tumour of myeloid origin that overrides the unusual and aberrant antigen expression (CD79a, CD99).\n\nIn conclusion, we considered, that a large panel of antibodies must be used to establish the challenging diagnosis of myeloid sarcoma. This condition that should always be considered as a diagnostic possibility in patients with MDS, AML or chronic myeloproliferative disorders complicated with a secondary tumor mass.\n\n\nConsent\n\nWritten informed consent was obtained from the patient for the management and publication of clinical and laboratory data. The Ethical Committee of the Hospital approved the publication of the clinical details (no. 780/14-01-2016).", "appendix": "Author contributions\n\n\n\nDemian S. managed the diagnostic and therapeutic procedures, provided details of clinical data, Horvath E performed data analysis and established the histopathological diagnosis, Nagy EE participated in data analysis, integration and performed the critical review of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nYilmaz AF, Saydam G, Sahin F, et al.: Granulocytic sarcoma: a systematic review. Am J Blood Res. 2013; 3(4): 265–270. PubMed Abstract \| Free Full Text\n\nJoseph JR, Wilkinson DA, Bailey NG, et al.: Aggressive Myeloid Sarcoma Causing Recurrent Spinal Cord Compression. World Neurosurg. 2015; 84(3): 866.e7–866.e10. PubMed Abstract \| Publisher Full Text\n\nHu X, Shahab I, Lieberman IH: Spinal Myeloid Sarcoma “Chloroma” Presenting as Cervical Radiculopathy: Case Report. Global Spine J. 2015; 5(3): 241–246. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDock G, Warthin A: A new case of chloroma with leukemia. Trans Assoc Am Phys. 1904; 19: 64–115.\n\nBakst RL, Tallman MS, Douer D, et al.: How I treat extramedullary acute myeloid leukemia. Blood. 2011; 118(14): 3785–3793. PubMed Abstract \| Publisher Full Text\n\nSeifert RP, Bulkeley W 3rd, Zhang L, et al.: A practical approach to diagnose soft tissue myeloid sarcoma preceding or coinciding with acute myeloid leukemia. Ann Diagn Pathol. 2014; 18(4): 253–60. PubMed Abstract \| Publisher Full Text" }	[ { "id": "12783", "date": "14 Mar 2016", "name": "Judit Csomor", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe main aim of this short case report is to create attention for myeloid sarcoma, which can be a difficult differential diagnostic problem, first of all in cases, when the myeloid sarcoma precedes the bone marrow disease. The documentation of the presented patient illustrates well the histological work up and the diagnosis, but the paper would have been more teaching if the authors add more detail about the patients course. I would have been interested in the results of investigation of the cerebrospinal fluid, the cytogenetic findings and the neurological status after operation? Why monotherapy was chosen? What further complications did they observe? What was the cause of death?In my experience myeloid sarcoma can be purely blastic, but frequently shows signs of myeloid differentiation. Fine cytoplasmic granulation of the immature cells, presence of eosinophilic myelocytes among the blasts, or some monocytoid features are suspicious for myeloid tumor, and can be recognised by careful examination of high quality sections and special stains. If these signs are observed a short panel containing the lineage specific markers is enough to confirm the diagnosis For differential diagnosis of a poorly differentiated tumor, the authors list a wide panel of immune stains emphasizing markers of immaturity (CD34, CD117, tdt, CD99). I think the lineage specific markers, especially CD45, MPO, CD3, CD79, Pax5 and lysozyme, are more important. To suggest a cost effective algorithm in the report would be a valuable supplement.And I have to add, that the diagnosis of MDS with excess of blasts and AML is first of all based on bone marrow smears and flow cytometry. If flow cytometry is not available cytochemistry is recommended for defining the cell lineage of the blasts. Bone biopsy is usually only an additional investigation (except of dry tap). Probably in the presented case smears also were elaborated, but data of smears and bone marrow biopsy is not clearly separated. The blast count is based on CD34 and CD117 immunohistochemistry without mentioning the ratio of the blast by marrow smear cytology. The authors state, that CD56 may have role in homing of the myeloid blasts, therefore additional CD56 investigation would be interesting. AML with t(8;21) is more frequently cause myeloid sarcoma, therefore applying FISH probes to test the genotype of the tumor is also recommended to get more information about these rare tumors.", "responses": [] }, { "id": "12978", "date": "22 Mar 2016", "name": "Erika Toth", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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-structured paper. Rare case, nice demonstration of the difficult differential diagnostic process of myeloid sarcoma. The title is appropriate and represent the work. Results supports the final conclusion of the case.", "responses": [] } ]	1	https://f1000research.com/articles/5-128
https://f1000research.com/articles/5-119/v1	29 Jan 16	{ "type": "Review", "title": "Catalysts of plant cell wall loosening", "authors": [ "Daniel J. Cosgrove" ], "abstract": "The growing cell wall in plants has conflicting requirements to be strong enough to withstand the high tensile forces generated by cell turgor pressure while selectively yielding to those forces to induce wall stress relaxation, leading to water uptake and polymer movements underlying cell wall expansion. In this article, I review emerging concepts of plant primary cell wall structure, the nature of wall extensibility and the action of expansins, family-9 and -12 endoglucanases, family-16 xyloglucan endotransglycosylase/hydrolase (XTH), and pectin methylesterases, and offer a critical assessment of their wall-loosening activity", "keywords": [ "plant cell wall", "cell wall expansion", "wall loosening" ], "content": "Introduction\n\nThe growing cell wall of plants is both strong and extensible. Its mechanical strength lets it resist the tensile stresses in the plane of the wall (~10 MPa or more) generated by the internal hydrostatic pressure (turgor) typical of plant cells (~0.5–1 MPa). Its extensibility lets it expand irreversibly in surface area by 10- to more than 1,000-fold between its initial formation at cell division and the subsequent cessation of growth at developmental maturity. Such expansion involves selective wall loosening to enable irreversible extension, or “creep” (see Table 1 for explanations of biomechanical terms; see 1 for additional details of the biomechanical aspects of plant cell growth). This process enables plant cells to grow to more than 100 times the size of their meristem initials. Lacking such a process, the tallest trees on Earth would be shorter than the average reader of this article. Synthesis and incorporation of new structural components into the growing walls are also required in the long term to prevent loss of mechanical integrity, but wall synthesis in most plant cells is not linked mechanistically to expansion, as it is in bacteria. In this article, I briefly summarize current concepts of plant cell wall loosening and the proteins that catalyze it.\n\n*This operational definition hides the fact that the irreversible component of strain for plant cell walls is complex and time-dependent, and may include a delayed elastic component and a viscous component as well as a plastic component. Plasticity is generally defined as rapid and irreversible deformation when stress exceeds a threshold. However, technical definitions of plasticity have varied among authors. See 1 for additional details.\n\nThe term “wall loosening” has been used in diverse contexts: for instance, the indiscriminate breakdown of wall polymers by ammonia explosion pretreatment of biomass for biofuel production2 and the oxidative scission of polysaccharides by hydroxyl radicals during seed germination, fruit softening, abscission, and defense responses3–8. Although one might first think of lytic actions as causing wall loosening, it turns out that the most potent of the natural wall-loosening catalysts—expansins—lack detectable wall lytic activity, presenting continuing enigmas about how they function at the molecular level and how the plant cell wall is structured to enable expansin-mediated wall loosening and surface expansion.\n\n\nEvolving concepts of cell wall structure\n\nThe growing cell wall is made of strong, stable, and inextensible cellulose microfibrils embedded in a hydrated matrix of polysaccharides classified as pectins and hemicelluloses9–12. Diverse proteins and proteoglycans are also present in small amounts. Concepts of how these components form a strong yet extensible wall have evolved considerably, inevitably influencing our notions of wall loosening. Fifty years ago, the growing cell wall was viewed as a mat of cellulose microfibrils embedded in an amorphous matrix that yielded plastically to the forces of cell turgor13,14. In this concept, wall loosening was thought to result from reduction of matrix viscosity by the action of lytic enzymes, but later results showed that changes in wall viscoelastic properties are not the basis for cell wall loosening and growth, at least in many contexts15,16. Nevertheless, these oversimplified notions of wall structure and wall loosening continue to exert a strong influence on current thinking.\n\nA major conceptual departure from this view came from the Albersheim group, who proposed that cellulose microfibrils were separated from each other by a massive macromolecule made of pectin, glycoprotein, and xyloglucan, with xyloglucan binding tightly to cellulose surfaces, thereby forming a load-bearing molecular network17. Such a structure would have very different mechanical properties than the previous concept of the cell wall as a fiberglass-like structure, and suggested potential sites and mechanisms for wall loosening. When these were not substantiated, the Albersheim model was abandoned in favor of an alternative in which xyloglucan directly tethered cellulose microfibrils to form an interconnected network that was embedded in a viscous, gel-like pectin matrix (Figure 1A)9,10,18. This “tethered network” model puts xyloglucan in the limelight as the major target of wall loosening and has dominated discussion of primary cell walls for more than two decades. It differs from the old model of an amorphous matrix reinforced with cellulose microfibrils in that xyloglucans were viewed as direct tethers that bind tightly and extensively to cellulose surfaces, forming the sole load-bearing links between cellulose microfibrils.\n\n(A) The tethered network model proposes that cellulose microfibrils (red) are well separated by matrix polysaccharides, including xyloglucans (blue) which bind to cellulose microfibrils and tether them to form a load-bearing molecular network. (B) The “biomechanical hotspot” model posits limited cellulose-cellulose junctions that are bonded together by a xyloglucan-cellulose amalgam (green) with limited enzymatic accessibility. The limited frequency of these junctions means that mesoscale aspects of wall architecture and motions may predominate over nanoscale structure in limiting cell enlargement. Additionally, xyloglucan is shown in both a coiled configuration and a highly extended form, but which form predominates in cell walls is uncertain.\n\nRecent results, however, have weighed against the tethered network model: (a) Arabidopsis mutants lacking xyloglucan have a relatively minor growth phenotype19–21, showing that xyloglucan is not essential for a functional, growing cell wall. (b) Nuclear magnetic resonance (NMR) results showed that xyloglucan-cellulose interactions are not as prevalent as expected from the model22, but that pectin-cellulose interactions are much more abundant than expected23,24. (c) Digestion of cell walls with xyloglucan-cutting enzymes did not reduce wall strength or cause cell wall extension, despite the prediction of the tethered network model25,26.\n\nA revised concept of wall structure emerged from a study that made use of the method outlined in Figure 2 to test the ability of substrate-specific endoglucanases to induce cell wall creep26. Enzymes that cut only xyloglucan or only cellulose did not induce cell wall creep, whereas endoglucanases able to cut both xyloglucan and cellulose did induce creep. A family-12 glycosyl hydrolase (GH12) named Cel12A, from the fungus Trichoderma reesei, was particularly effective at causing cell wall creep. Enigmatically, the combination of xyloglucan-specific and cellulose-specific enzymes—both GH12 enzymes and structurally similar to Cel12A—lacked wall-loosening action. This puzzling result was interpreted to mean that walls were loosened only when a relatively inaccessible amalgam containing xyloglucan and cellulose was digested by a single enzyme with both xyloglucanase and cellulase activities. To account for the ineffectiveness of two separate enzymes with distinct substrate specificities, the amalgam was hypothesized to be buried within tight junctions between two or more cellulose microfibrils. These and other results led to the revised concept depicted in Figure 1B, in which wall extensibility is controlled at limited sites (“biomechanical hotspots”) of close contact between cellulose microfibrils26.\n\n(A) A cell wall sample is prepared from a growing plant tissue, such as a young hypocotyl from a seedling, and clamped at constant force in an apparatus that continuously measures changes in sample length. The buffer surrounding the sample can be exchanged for one containing a candidate wall-loosening protein. (B) Time course for change in length, using a typical response to α-expansin as an example. The cell wall creep measured in this device is dependent on continuous wall loosening by expansins or other proteins, and thus mimics aspects of cell wall enlargement in living cells.\n\nSubsequent results support the concept that cellulose-cellulose contacts may be important for wall mechanics. Making use of advances in atomic force microscopy (AFM), studies of never-dried primary cell walls showed the nanoscale arrangement of cellulose microfibrils and the presence of cellulose-cellulose junctions27,28. The ability to image cell walls under water is a key advantage of AFM compared with high-resolution scanning electron microscopy, which requires the sample to be dry, potentially causing wall polymers to coalesce. Water plays a big role in the structure and mechanics of primary cell walls29–31. Other recent work used molecular dynamics simulations to show that cellulose-cellulose junctions, glued together by a monolayer of xyloglucan, are strong enough to contribute substantially to cell wall mechanics32. A clue to the potential role of the bulk of xyloglucans in the wall emerged from a recent study of an Arabidopsis mutant lacking xyloglucan: cellulose microfibrils were parallel to each other, whereas in the wild type they were more dispersed21. This result suggests that xyloglucans may orchestrate cellulose-cellulose interactions in complex ways.\n\nThe revised model in Figure 1B does not address the potential role of direct pectin-cellulose interactions24. NMR results show that pectins include both mobile and rigid chains23, interpreted to mean that some pectins form a mobile gel-like milieu but that others are tightly associated with cellulose. The latter component may contribute to the cellulose-cellulose junctions or may provide a separate set of linkages between cellulose microfibrils29,33. The extent of pectin-cellulose cross-peaks in NMR cross-polarization experiments implies an interaction that is more stable than that detected by in vitro binding experiments34, but does not demonstrate it to be load-bearing. This remains an unresolved aspect of cell wall structure. How tensile forces in the wall are transmitted between cellulose microfibrils is a key question for understanding the molecular mechanism of wall loosening because these are the connections that must be loosened for the wall to expand irreversibly. The biomechanical hotspot concept proposes that growing cell walls contain specific, built-in junctions designed for slippage and stress relaxation by the action of expansins and other wall-loosening proteins.\n\nIn biophysical terms, cell growth begins by selective loosening of the cell wall, resulting in a relaxation of wall stress; this action creates the impetus for water uptake and physical enlargement of the cell by a process in which the wall polymers slide or otherwise separate to increase wall surface area1,35. Wall loosening has been studied in vitro by measuring sustained cell wall extension (creep) with an extensometer, sketched in Figure 236. Such cell wall creep mimics the sustained enlargement of cell walls during plant growth, and enabled the discovery and initial characterization of expansins37,38. This approach reduces the complexity inherent in living cells, which may manipulate wall pH, redox state, and other variables by dynamic signaling pathways39.\n\nA legion of lytic enzymes—from plants as well as from pathogens—can cleave the backbone or sidechains of wall polysaccharides, and may even digest the cell wall to the point of mechanical failure. Such enzymatic deconstruction, potentially aided by the chemical action of hydroxyl radicals, may contribute to fruit softening, organ abscission, and pathogen attack, but these lytic activities generally do not cause sustained cell wall creep40,41. Evidently, wall loosening during cell enlargement is subtler than simple breakdown of cell wall polymers. The rest of this review summarizes the action of enzymes and other wall-active proteins ascribed a wall-loosening function. The complex actions of reactive oxygen species, such as hydroxyl radicals42–45, are beyond the scope of this review. The first group of wall-loosening proteins to be discussed (expansins) have no detectable enzymatic activity, yet are the clearest examples of endogenous catalysts of plant cell wall loosening. The term “catalyst” is used here in the general sense and does not imply a change in the covalent structure of cell wall components.\n\n\nExpansins\n\nThe activity of three classes of expansins has been characterized to date: α-expansins, β-expansins, and bacterial expansins46,47. The first expansins—now identified as α-expansins—were discovered by a reconstitution approach in which protein extracts from growing plant cell walls were added to heat-inactivated cell walls clamped in a extensometer to restore their ability to extend irreversibly (Figure 2)48. The proteins induced wall creep and wall relaxation, yet they neither hydrolyzed the cell wall nor exhibited other enzyme activities49–51. Their wall-loosening activity was maximal at low pH (~4), consistent with their role in the so-called acid growth response of plants and the rapid induction of cell elongation by auxin-induced acidification of the cell wall space52,53.\n\nExperiments with cucumber hypocotyl walls showed that α-expansins did not weaken the cell wall, as measured by mechanical (stress/strain) assays54. The ability of α-expansins to induce creep without reducing wall stiffness provided additional evidence that they do not cut cell wall linkages, which would result in reduced cell wall stiffness as well as release of wall polysaccharide fragments. Other studies showed that α-expansin binding to matrix-depleted cell walls saturated at a value of approximately 1:1,000 (dry mass of protein:wall)50. A recent calculation showed that this value corresponds to a spacing of approximately 200 nm between expansin binding sites within a cell wall lamella55, implying that the mesoscale (between molecular and cellular scales56) is the appropriate scale for understanding the mechanics of growing cell walls. Molecular-scale models of cell walls (i.e., a 50-nm cube) may be focused on too small a piece of the cell wall to capture crucial structural aspects of cell wall growth.\n\nApplication of α-expansins to living tobacco cell cultures enhanced cell growth57, consistent with a host of reports in which ectopic expression of α-expansin genes likewise stimulated plant growth (reviewed in 38,58). Wang et al.59 used a single-cell compression assay to estimate the stiffness (elastic modulus) and the bursting force of living tomato suspension culture cells treated with α-expansin. Within a physiologically realistic extracellular pH range (4.5–6.0), α-expansin treatment did not change wall stiffness, consistent with the results cited above for cucumber hypocotyls54. Curiously, a higher force was required to cause bursting of cells treated with α-expansin at acidic pH values (when α-expansins are most active), compared with untreated cells. The increased toughness may be a consequence of enhanced force dissipation by α-expansin-mediated wall relaxation during the compression, evidenced by higher strain at failure for cells treated with α-expansin. Apparently, the loosening action of α-expansin can result in what appears to be a tougher wall (greater mechanical energy required for failure). A lesson from this example is that different mechanical assays report on different aspects of cell wall mechanics, and what may seem at first glance to be a contradictory result may be consistent with a specific loosening mechanism.\n\nContrary to the above reports that α-expansin does not mechanically weaken cell walls, constitutive overexpression of an α-expansin gene in rice suspension cells resulted in a six-fold reduction in wall stiffness as measured by micro-indentation assay60. It would be premature, however, to conclude that such weakening was a direct action of α-expansin, because large differences in cell size and wall composition were noted between control and the constitutive overexpressor cell lines, and because it is likely that α-expansin overexpression led to changes in wall synthesis and assembly that impacted the micro-indentation results.\n\nA second set of plant expansins encompasses the β-expansin group, also encoded by a multigene family throughout land plants38. Characterization of protein activity has been limited almost exclusively to a unique clade of β-expansins that are expressed at high levels in grass pollen61–64, and that were evolutionarily co-opted in grasses to aid penetration of the pollen tube through the grass stigma and style65,66. These proteins have drawn attention in the immunology field because they are major allergens of grass pollen; thus, their alias as “group I grass pollen allergens”67. The crystallographic structure of β-expansin from maize pollen revealed a two-domain protein with domain one (D1) resembling the fold of family-45 endoglucanases (GH45 in the www.cazy.org classification system) and a second domain (D2) forming a β-sandwich with a presumptive binding function64. It is notable that some of the GH45 catalytic residues are conserved in plant expansins, but a key aspartic acid residue that functions as the general base in many GH45 endoglucanases is missing, potentially accounting for the lack of hydrolytic activity.\n\nThe β-expansins in the pollen-allergen group have two properties that may not be common to the larger group of β-expansins: they selectively loosen cell walls of plants in the grass family (Poaceae), which have a wall composition distinctive from that of most land plants63, and they solubilize matrix polysaccharides—arabinoxylan and homogalacturonan (HG)—found both in the cell wall and in the intercellular adhesive, or middle lamella, between cells of grasses68. Solubilization of the matrix suggested a lytic action, but several tests for lytic activities gave negative results. It is relevant here to note that these polymers can be solubilized from walls by chemical extractants that do not break covalent bonds. Unlike α-expansins, pollen β-expansins greatly reduced the tensile strength of grass cell walls, at least in part by weakening the middle lamella between cells, whereas they had negligible effect on cell walls from eudicot species69. These results suggest that eudicot walls lack the specific target of pollen β-expansins, or that the target has a minor mechanical role in eudicot walls. Binding studies suggested arabinoxylan (a hemicellulose) as a potential binding target64; however, not all cell walls rich in arabinoxylan were loosened by pollen β-expansin63, leading to the suggestion that grass cell walls have a unique cross-linking structure that is the specific target of pollen β-expansins. Further work is needed to identify this wall component and its structural role in grass cell walls. Moreover, the loosening actions of other β-expansins, outside the pollen group, have not yet been explored, in part because they have been difficult to extract from cell walls in active form70 and because attempts to produce plant expansin proteins by heterologous expression have met little success.\n\nA third group—bacterial expansins—was recognized through structural and phylogenetic approaches. The crystal structure of a Bacillus subtilis protein, renamed BsEXLX1 according to expansin nomenclature, was found to be homologous to the structure of pollen β-expansin71. Wall extension assays showed that BsEXLX1 could induce cell wall creep, but only weakly. Like α-expansins, it did not weaken cell walls in stress/strain assays nor did it exhibit lytic activity with isolated cell wall polysaccharides or with whole cell walls as substrates, yet it weakened paper, a mat of pure cellulose fibers. In these respects, it behaved like a weak α-expansin.\n\nPhylogenetic analysis identified expansins in a number of other bacteria that are plant pathogens72,73, evidently the result of horizontal gene transfer from plants. When expansins from Xanthomonas campestris, Clavibacter michiganensis, Ralstonia solanacearum, and Aspergillus niger (all plant pathogens) were recombinantly expressed in Escherichia coli and tested for their ability to induce creep of cell walls, they consistently exhibited positive but weak activity74. Gene knockout experiments, recently reviewed46, indicate that bacterial expansins facilitate bacterial colonization of plant surfaces. Exactly how this works is unclear because their wall-loosening activity is so weak; their specific activity is perhaps 100 times lower compared with α-expansins. We do not understand the structural basis for the high activity of α-expansins versus low activity of bacterial expansins, but the consistently low activity of the latter may have evolved to avoid plant defenses that sense cell wall integrity75,76.\n\nBecause plant expansins have proven so difficult to express in active recombinant form, the B. subtilis expansin BsEXLX1 was used in place of plant expansins for extensive structure-function analysis by site-directed mutagenesis77, crystallography78, and NMR79, combined with binding and activity assays (Figure 3). We have learned a lot from these studies. Domain D2 proved to be the major determinant of expansin binding to plant cell walls, but two distinctive modes of binding to cellulose and to pectin were identified. Site-directed mutagenesis showed that binding to cellulose required three aromatic residues on the surface of D277. This was confirmed and extended by crystallographic studies of protein-ligand complexes78, which showed that these three residues bound alternating glucose residues in cellulose oligosaccharides, predominantly through hydrophobic interactions (Figure 3). Cellulose binding was required for wall loosening. On the other hand, binding of BsEXLX1 to whole cell walls was dominated by electrostatic binding to acidic polysaccharides via non-conserved basic residues on the “back side” of the D2 domain. Mutagenesis of these basic residues greatly reduced total wall binding, but actually increased wall creep activity77 and enhanced binding to cellulose within the wall, as detected by 13C solid-state NMR79. Domain D1 did not bind to cellulose or whole cell walls yet was essential for activity. Site-directed mutagenesis also showed that an aspartic acid residue (Asp82) in D1 was essential for creep activity; this residue is part of the catalytic site conserved in GH45 endoglucanases and MltA-type lytic transglycosylases71. This result suggests a cryptic enzymatic activity that has yet to be discovered. Pastor et al.80 hypothesized, as an alternative idea, that electrostatic polarization on the expansin surface may mediate its wall-loosening action by weakening hydrogen bonding within cellulose. In an NMR study of BsEXLX1 targeting within complex plant cell walls, expansin was seen to bind cellulose with a different chemical shift than bulk cellulose, indicating a slightly modified configuration of the glucan chains in the cellulose target79. Whether this modification was a result of expansin action is uncertain; more likely, expansin selectively binds to an altered form of cellulose. Moreover, xyloglucan was in close proximity to the binding site, which thus resembled the biomechanical hotspots described above. The lessons learned from structure-function analysis of bacterial expansin extend in part to plant expansins, but functional differences still lack structural explanations (e.g., why bacterial expansins are less active than α-expansin, and why they lack the matrix-solubilizing activity of the pollen β-expansins).\n\nTwo proteins (red and blue) in the crystallographic unit form a sandwich-like structure with cellohexaose (green), an oligosaccharide form of cellulose78. The interactions with cellohexaose are mediated exclusively through the open planar surface of the second domain (D2) and depend mostly on hydrophobic interactions with three aromatic residues arranged in a spaced, linear configuration so they bind the hydrophobic face of alternating glucose residues. The sandwich-like structure probably does not form in cell walls, but it provides structural information about the interaction of expansin with cellulose surfaces. Abbreviations: D1, domain 1; D2, domain 2.\n\nBsEXLX1 and other bacterial expansins have also drawn considerable attention as possible synergists of cellulose deconstruction by cellulases, with contradictory reports. This topic was recently reviewed46, and the conclusion was that the reported synergistic actions of BsEXLX1 addition were attributable, at least in part, to non-specific protein effects that predominate at very low cellulase loadings and low cellulose conversion (~1%). To be relevant for commercial use, synergistic activity at high cellulose conversion should be demonstrated.\n\nIn summary, the three classes of expansins outlined here are similar in their two-domain structure and their ability to induce creep of plant cell walls, but their biological roles differ:\n\na. α-expansins mediate acid-induced extension of plant cell walls without mechanically weakening the cell walls;\n\nb. pollen β-expansins not only cause cell wall creep but also solubilize polysaccharides in the middle lamella between the cell walls of grasses (but not other plants), thereby facilitating penetration of the pollen tube to the ovary; the physical actions of other β-expansins are almost certainly different but have yet to be documented;\n\nc. bacterial expansins facilitate colonization of plant tissues by a mechanism yet to be established but presumably linked to their weak wall-loosening action.\n\n\nEndoglucanases and endotransglucosylases\n\nThese two classes of plant enzymes are often called wall-loosening enzymes, a point to be examined below, but first it is instructive to compare the loosening action of α-expansin with that of the fungal endoglucanase Cel12A, described above. Wall creep induced by Cel12A begins after a substantial lag, 6 to more than 60 minutes, depending on enzyme concentration54, whereas with α-expansin it begins within seconds37. Cel12A treatment increased the elastic and plastic compliances of cucumber hypocotyl walls, but α-expansin treatment did not. Cel12A hydrolyzed xyloglucan and cellulose, releasing fragments to the buffer, but this was not the case for α-expansin. It is possible that both of these proteins exert their wall-loosening effects at the same sites (biomechanical hotspots) but by different mechanisms: α-expansin induces slippage at these junctions, whereas Cel12A digests the junctions. But is there evidence that plant enzymes possess wall-loosening activity similar to that of Cel12A?\n\nAccording to genome analyses as well as enzymatic assays, plants possess diverse wall lytic enzymes classified into numerous families41,81, two of which (GH9 and GH16) may cut β1,4-D-glucans (e.g., xyloglucan or cellulose). In the plant cell wall literature, GH16 enzymes are usually called xyloglucan endotransglucosylase/hydrolases (XTH) and are encoded by a large multigene family82,83. They cut xyloglucan and join the new reducing end to the non-reducing end of another xyloglucan (a transglucosylation) or to water (a hydrolysis). When XTH enzymes were first discovered, they were hypothesized to be wall-loosening enzymes, but subsequent experiments show them to have little or no ability to induce cell wall creep and to exert only minor effects on wall mechanics (assessed with stress/strain measurements). For instance, an XTH with strong transglucosylase activity from tomato was tested for its ability to induce wall creep or to weaken the wall as measured in stress/strain assays, with negative results25. These results are consistent with results obtained with xyloglucan-specific GH12 endoglucanases26, described above, showing that cutting of xyloglucan is not sufficient for cell wall loosening. Endogenous XTH activity in Arabidopsis hypocotyls is highest after elongation ceases84, indicating a turnover or remodeling function other than wall loosening. Genetic knockout of XTH genes expressed in the Arabidopsis root and hypocotyl completely eliminated xyloglucan hydrolase activity, but did not result in a growth phenotype85, indicating a dispensable role for growth. Consistent with this conclusion, transgenic overexpression of XTH in tomato hypocotyls did not affect hypocotyl growth86 but did result in subtle changes in wall composition, accompanied by small (<5%) and inconsistent changes in mechanical extensibility. When four different XTH genes were constitutively overexpressed in Arabidopsis, small (~10%) increases in hypocotyl length were observed for two genes, but no effect was observed for the other two genes. In contrast, Van Sandt et al.87 concluded that a recombinant Selaginella XTH caused wall loosening when applied to onion walls, but the effects were small. They observed a mechanical effect of XTH application when force was applied in the direction transverse to net cellulose orientation, but not in the direction parallel to net cellulose orientation. Their assay involved measuring wall extension immediately upon application of force; walls treated with exogenous XTH extended more rapidly than control walls during a time window of 10–30 minutes after application of the force. This result suggests that dynamical remodeling of xyloglucans in a rapidly extending wall may synergistically enhance wall extension, but it does not show that XTH itself can induce wall relaxation or creep. With this mode of action, XTH might be termed an indirect or secondary loosening agent88 to differentiate its indirect action from that of a primary loosening agent that directly catalyzes cell wall creep.\n\nSimmons et al.89 recently reported that an enzyme in the GH16/XTH family, uniquely found in horsetail (Equisetum spp.), was able to carry out an unusual transglucosylation, using cellulose as the lytic (donor) substrate and xyloglucan as the acceptor substrate. This action would be expected to form a covalent link between cellulose and xyloglucans and might result in cell wall stiffening. However, the mechanical consequences of this unusual GH16 activity have not been reported. An XTH enzyme from barley also showed activity with cellulose-like substrates, but the activity was very low90.\n\nTo summarize this section, the totality of GH16 results leads me to conclude that XTH does not cause appreciable cell wall loosening, but is likely involved in xyloglucan remodeling and turnover during primary wall formation and after cell elongation has ceased. Some GH16 enzymes may stitch newly synthesized xyloglucan chains into xyloglucans already anchored in the wall, thus forming larger molecules55,91. Why such action has so little effect on wall mechanics seems puzzling—an indication that we lack a deep understanding of the structural determinants of plant cell wall mechanics.\n\nLet us now consider plant GH9 enzymes, often called endoglucanases or cellulases. The database at www.cazy.org identifies a variety of activities for (mostly microbial) GH9 enzymes, including hydrolysis of xyloglucans, mannans, and xylans, as well as cellulose and (1,3;1,4)-β-D-glucans, so it is possible that the plant enzymes do more than cut cellulose or xyloglucan. This is consistent with their sequence diversity: phylogenetic analysis revealed more than 11 diverse GH9 clades in plants, and expression patterns indicate that they are involved in cell wall modification during fruit softening, abscission, growth, wood formation, and defense92–96. One well-studied GH9 clade includes a membrane-associated endoglucanase (called KORRIGAN) that is part of the cellulose synthesis complex and that influences the organization of cellulose in the wall97–99.\n\nWhether plant GH9 enzymes directly cause wall relaxation and expansion is uncertain, but limited experimental results support this possibility. Two studies reported that plant GH9 enzymes can hydrolyze cellulose and xyloglucan in vitro100,101, so they may be able to induce cell wall creep, but this test has not been reported. In contrast to these two reports, another GH9 enzyme from tomato could cut (1,3;1,4)-β-D-glucan but was unable to cut either xyloglucan or crystalline cellulose102. Overexpression of a poplar GH9 gene in Arabidopsis resulted in high levels of cello-oligosaccharides in the leaf, taken as evidence of cellulase action by the enzyme103. Leaf growth was increased in the overexpressing lines, as was plastic compliance, measured in stress/strain assays.\n\nIn summary, the limited experimental results suggest that some plant GH9 enzymes may directly loosen the cell wall to induce stress relaxation and wall creep, but more work is needed to demonstrate direct wall-loosening activity and to test whether plants actually use these enzymes for this function in vivo.\n\n\nPectin methylesterase and other pectin-modifying enzymes\n\nInterest in the potential wall-loosening activity of pectin-modifying enzymes has increased recently104–106, in part because of puzzling results suggesting that sites of leaf initiation on shoot apical meristems are softer (lower elastic modulus) as a result of de-esterification of pectin (HG)107,108. HG is synthesized in the Golgi apparatus and delivered to the cell wall with most of the carboxyl groups blocked with methyl esters12, making it resistant to the lytic action of pectate lyase and many endogalacturonases. Disruption of the normal delivery of pectin to the cell wall109, or its de-esterification106, leads to substantial growth defects. From studies of nuclear magnetic spin transfer within Arabidopsis cell walls22,110 and mechanical assays of Arabidopsis pectin mutants111, it appears that pectins are physically entangled with xyloglucan within the wall matrix. After delivery of HG to the cell wall, methyl esters are removed by the action of pectin methylesterase (PME), encoded in plants by a large multigene family. The puzzle mentioned above stems from the contradiction with well-established results showing that de-esterified pectins in vitro form stiffer gels than do methyl-esterified pectins, and that pectin de-esterification in vivo is associated with cell wall stiffening as cells cease elongation112,113. Stiffening arises from cooperative calcium binding of contiguous carboxyl groups on two adjacent pectin chains114. Thus, one would expect that regions of de-esterified HG in the meristem would be stiffer, not softer. Contrary to this expectation, reduction of PME activity by ectopic expression of PME inhibitor proteins resulted in stiffer walls, measured by micro-indentation of the plant surface106–108. At this point, the reason for the softer walls in regions rich in de-esterified pectin is unexplained. One possibility is that de-esterified pectins get cleaved into shorter chains by endogenous endogalacturonase and lyase, but there is scant evidence for this. Another possibility is that walls in the meristem lack sufficient calcium for pectic gel formation; without calcium cross-linking, the negative charges on the HG chains might cause cell wall swelling and softening. A third possibility is that manipulation of the state of pectin activates cell wall integrity sensors, activating brassinosteroid signaling115 and potentially inducing many changes in cell wall composition and structure that result in altered wall mechanics. Further work will be necessary to understand these contrary associations between pectin esterification and wall stiffness. In any case, there is no evidence that PME directly causes wall stress relaxation or creep, so its action is of a different kind altogether.\n\n\nProspectus\n\nRecent studies are converging on the concept that the primary cell wall contains limited cellulose-cellulose junctions that are sites of initial wall loosening and stress relaxation, and that are the selective targets of expansins and potentially other wall-loosening agents. How these sites are formed is unknown; are they the result of a well-controlled cellular process or of a purely physical, stochastic interaction? Their detailed structure and spatial distribution need to be investigated, perhaps starting with novel tagging procedures. We also need to know whether plant GH9 enzymes can loosen the wall in the manner of Cel12A. The contradictory reports of PME action on cell wall properties present an unresolved puzzle, and the functional significance of extensive pectin-cellulose interactions, seen in NMR studies, needs deeper study to understand their possible significance for cell wall mechanics and growth.\n\n\nAbbreviations\n\nAFM, atomic force microscopy; D1, expansin domain 1; D2, expansin domain 2; GH9, glycosyl hydrolase family 9; GH12, glycosyl hydrolase family 12; GH16, glycosyl hydrolase family 16; GH45, glycosyl hydrolase family 45; HG, homogalacturonan; NMR, nuclear magnetic resonance; PME, pectin methylesterase; XTH, xyloglucan endotransglycosylase/hydrolase.", "appendix": "Competing interests\n\n\n\nThe author declares that he has no competing interests.\n\n\nGrant information\n\nWork on expansins was supported by US Department of Energy Grant DE-FG02-84ER13179 from the Physical Bioscience Program, Office of Basic Energy Sciences. Work on wall structure was supported as part of the Center for Lignocellulose Structure and Formation, an Energy Frontier Research Center funded by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under Award Number DE-SC0001090.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nCosgrove DJ: Plant cell wall extensibility: connecting plant cell growth with cell wall structure, mechanics, and the action of wall-modifying enzymes. J Exp Bot. 2016; 67(2): 463–76. PubMed Abstract \| Publisher Full Text\n\nPattathil S, Hahn MG, Dale BE, et al.: Insights into plant cell wall structure, architecture, and integrity using glycome profiling of native and AFEXTM-pre-treated biomass. J Exp Bot. 2015; 66(14): 4279–94. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMiedes E, Suslov D, Vandenbussche F, et al.: Xyloglucan endotransglucosylase/hydrolase (XTH) overexpression affects growth and cell wall mechanics in etiolated Arabidopsis hypocotyls. J Exp Bot. 2013; 64(8): 2481–97. PubMed Abstract \| Publisher Full Text\n\nKaewthai N, Gendre D, Eklöf JM, et al.: Group III-A XTH genes of Arabidopsis encode predominant xyloglucan endohydrolases that are dispensable for normal growth. Plant Physiol. 2013; 161(1): 440–54. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nMiedes E, Zarra I, Hoson T, et al.: Xyloglucan endotransglucosylase and cell wall extensibility. J Plant Physiol. 2011; 168(3): 196–203. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nVan Sandt VS, Suslov D, Verbelen JP, et al.: Xyloglucan endotransglucosylase activity loosens a plant cell wall. Ann Bot. 2007; 100(7): 1467–73. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nCosgrove DJ: Enzymes and other agents that enhance cell wall extensibility. Annu Rev Plant Physiol Plant Mol Biol. 1999; 50: 391–417. PubMed Abstract \| Publisher Full Text\n\nSimmons TJ, Mohler KE, Holland C, et al.: Hetero-trans-β-glucanase, an enzyme unique to Equisetum plants, functionalizes cellulose. Plant J. 2015; 83(5): 753–69. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nHrmova M, Farkas V, Harvey AJ, et al.: Substrate specificity and catalytic mechanism of a xyloglucan xyloglucosyl transferase HvXET6 from barley (Hordeum vulgare L.). FEBS J. 2009; 276(2): 437–56. PubMed Abstract \| Publisher Full Text\n\nThompson JE, Smith RC, Fry SC: Xyloglucan undergoes interpolymeric transglycosylation during binding to the plant cell wall in vivo: Evidence from 13C/3H dual labelling and isopycnic centrifugation in caesium trifluoroacetate. Biochem J. 1997; 327: 699–708. Reference Source\n\nUrbanowitcz B: Glycoside Hydrolase Family 9/Plant endoglucanases. In: Wilson DB, editor. CAZYpedia, accessed 21 September 2015. Reference Source\n\nLibertini E, Li Y, McQueen-Mason SJ: Phylogenetic analysis of the plant endo-beta-1,4-glucanase gene family. J Mol Evol. 2004; 58(5): 506–15. PubMed Abstract \| Publisher Full Text\n\nUrbanowicz BR, Bennett AB, Del Campillo E, et al.: Structural organization and a standardized nomenclature for plant endo-1,4-beta-glucanases (cellulases) of glycosyl hydrolase family 9. Plant Physiol. 2007; 144(4): 1693–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBuchanan M, Burton RA, Dhugga KS, et al.: Endo-(1,4)-β-glucanase gene families in the grasses: temporal and spatial co-transcription of orthologous genes. BMC Plant Biol. 2012; 12: 235. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDu Q, Wang L, Yang X, et al.: Populus endo-β-1,4-glucanases gene family: genomic organization, phylogenetic analysis, expression profiles and association mapping. Planta. 2015; 241(6): 1417–34. PubMed Abstract \| Publisher Full Text\n\nMcNamara JT, Morgan JL, Zimmer J: A molecular description of cellulose biosynthesis. Annu Rev Biochem. 2015; 84: 895–921. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVain T, Crowell EF, Timpano H, et al.: The Cellulase KORRIGAN Is Part of the Cellulose Synthase Complex. Plant Physiol. 2014; 165(4): 1521–32. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nLei L, Zhang T, Strasser R, et al.: The jiaoyao1 Mutant Is an Allele of korrigan1 That Abolishes Endoglucanase Activity and Affects the Organization of Both Cellulose Microfibrils and Microtubules in Arabidopsis. Plant Cell. 2014; 26(6): 2601–16. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nOhmiya Y, Takeda T, Nakamura S, et al.: Purification and properties of wall-bound endo-1, 4-beta-glucanase from suspension-cultured poplar cells. Plant Cell Physiol. 1995; 36(4): 607–14. PubMed Abstract\n\nYoshida K, Komae K: A rice family 9 glycoside hydrolase isozyme with broad substrate specificity for hemicelluloses in type II cell walls. Plant Cell Physiol. 2006; 47(11): 1541–54. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nUrbanowicz BR, Catalá C, Irwin D, et al.: A tomato endo-beta-1,4-glucanase, SlCel9C1, represents a distinct subclass with a new family of carbohydrate binding modules (CBM49). J Biol Chem. 2007; 282(16): 12066–74. PubMed Abstract \| Publisher Full Text\n\nPark YW, Tominaga R, Sugiyama J, et al.: Enhancement of growth by expression of poplar cellulase in Arabidopsis thaliana. Plant J. 2003; 33(6): 1099–106. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nPeaucelle A, Braybrook S, Höfte H: Cell wall mechanics and growth control in plants: the role of pectins revisited. Front Plant Sci. 2012; 3: 121. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWolf S, Greiner S: Growth control by cell wall pectins. Protoplasma. 2012; 249(Suppl 2): S169–75. PubMed Abstract \| Publisher Full Text\n\nPeaucelle A, Wightman R, Höfte H: The Control of Growth Symmetry Breaking in the Arabidopsis Hypocotyl. Curr Biol. 2015; 25(13) 1746–52. PubMed Abstract \| Publisher Full Text\n\nPeaucelle A, Braybrook SA, Le Guillou L, et al.: Pectin-induced changes in cell wall mechanics underlie organ initiation in Arabidopsis. Curr Biol. 2011; 21(20): 1720–6. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nBraybrook SA, Peaucelle A: Mechano-chemical aspects of organ formation in Arabidopsis thaliana: the relationship between auxin and pectin. PLoS One. 2013; 8(3): e57813. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhu C, Ganguly A, Baskin TI, et al.: The fragile Fiber1 kinesin contributes to cortical microtubule-mediated trafficking of cell wall components. Plant Physiol. 2015; 167(3): 780–92. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDick-Pérez M, Zhang Y, Hayes J, et al.: Structure and interactions of plant cell-wall polysaccharides by two- and three-dimensional magic-angle-spinning solid-state NMR. Biochemistry. 2011: 50(6): 989–1000. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nAbasolo W, Eder M, Yamauchi K, et al.: Pectin may hinder the unfolding of xyloglucan chains during cell deformation: implications of the mechanical performance of Arabidopsis hypocotyls with pectin alterations. Mol Plant. 2009; 2(5): 990–9. PubMed Abstract \| Publisher Full Text\n\nGoldberg R, Morvan C, Roland JC: Composition, Properties and Localization of Pectins in Young and Mature Cells of the Mung Bean Hypocotyl. Plant Cell Physiol. 1986; 27(3): 417–29. Reference Source\n\nZhao Q, Yuan S, Wang X, et al.: Restoration of mature etiolated cucumber hypocotyl cell wall susceptibility to expansin by pretreatment with fungal pectinases and EGTA in vitro. Plant Physiol. 2008; 147(4): 1874–85. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMorris ER, Powell DA, Gidley MJ, et al.: Conformations and interactions of pectins. I. Polymorphism between gel and solid states of calcium polygalacturonate. J Mol Biol. 1982; 155(4): 507–16. PubMed Abstract \| Publisher Full Text\n\nWolf S, van der Does D, Ladwig F, et al.: A receptor-like protein mediates the response to pectin modification by activating brassinosteroid signaling. Proc Natl Acad Sci U S A. 2014; 111(42): 15261–6. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation" }	[ { "id": "12198", "date": "29 Jan 2016", "name": "A. Lacey Samuels", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12199", "date": "29 Jan 2016", "name": "Olivier Hamant", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-119
https://f1000research.com/articles/5-118/v1	29 Jan 16	{ "type": "Review", "title": "Recent Advances in the Diagnosis and Treatment of Clostridium Difficile Infection", "authors": [ "Meera B. Avila", "Nathaniel P. Avila", "Andrew W. Dupont", "Meera B. Avila", "Nathaniel P. Avila" ], "abstract": "Clostridium difficile infection (CDI) has become the most frequently reported health care-associated infection in the United States [1]. As the incidence of CDI rises, so too does the burden it produces on health care and society. In an attempt to decrease the burden of CDI and provide the best outcomes for patients affected by CDI, there have been many recent advancements in the understanding, diagnosis, and management of CDI. In this article, we review the current recommendations regarding CDI testing and treatment strategies.", "keywords": [ "Clostridium Difficile", "CDI", "antibiotics", "microbiota" ], "content": "Introduction\n\nClostridium difficile is an opportunistic organism that causes infection in patients with an alteration in intestinal microbiota. Microbiota is the community of organisms that inhabits a particular region of the body, and the intestine is composed of 300–500 species of bacteria. Alteration in intestinal microbiota predisposes patients to becoming infected with the spores from C. difficile via fecal-oral transmission2. Once a patient has C. difficile infection (CDI), outcomes can range from asymptomatic colonization to severe diarrhea. Fulminant or severe complicated CDI is characterized by inflammatory lesions and the formation of pseudomembranes in the colon, which can lead to toxic megacolon, bowel perforation, sepsis, shock, and death2. In addition, CDI has become nefarious for more severe disease associated with frequent recurrences despite appropriate and adequate treatment3, in part due to a virulent strain of CD termed NAP1/B1/0274. The consequences of CDI affect the patient and society alike, as more than 300,000 hospitalizations involve CDI each year. The mean cost of each hospitalization ranges from $8911 to $30,049 per patient, at a yearly cost estimated at $1.0 to $4.9 billion to the US health care system5,6. While a large portion of this cost is related to a true increase in CDI incidence, some of the cost burden can be attributed to the over-diagnosis of CDI after the introduction of molecular tests7–10. As health care costs rise, so does the importance of continued research in the detection and treatment of CDI.\n\nIn the molecular era, how to best diagnose CDI in a cost-effective manner has become an area of much debate. In order to efficiently and effectively treat CDI, the diagnosis should be made rapidly based on clinical and laboratory evidence of the infection. Testing for CDI should only occur if patients have clinical risk factors for the disease along with signs and symptoms, most commonly diarrhea11. The most common risk factors include patients who are currently receiving antibiotics or who have received antibiotics in the past 8 weeks12. There are compelling data that almost all antibiotics can increase the risk of CDI, but third-generation cephalosporins, clindamycin, amoxicillin, and fluoroquinolones have been the most frequently reported12–14. In addition, patients are at greater risk if their age is greater than 65, if they are hospitalized or were recently hospitalized, or if they live in long-term care facilities4.\n\nLaboratory testing for CDI should be performed only on symptomatic patients and only on diarrheal stool15–17. Additionally, testing patients with CDI for “cure or clearance” or for “colonization” after treatment is not appropriate and not recommended16. Treated patients often shed spores for several weeks to months despite being asymptomatic, and further testing can lead to inappropriate courses of treatment15,17. There is general consensus that radiologic diagnosis of CDI is of little value18; however, imaging should be done in cases of suspected toxic megacolon. Endoscopic diagnosis should be reserved for cases when a diagnosis is emergently needed, if there is delay in implementing CDI testing, if laboratory tests are negative and CDI is strongly suspected, or in cases of ileus when stool is unavailable19.\n\nLaboratory testing for CDI is an exciting and rapidly changing field; however, it remains an area of confusion, largely because there is no generally accepted gold standard or single best test20. In general, the clinical usefulness of a CDI diagnostic test is judged on its sensitivity, specificity, turnaround time (TAT), cost, and availability21. Currently, the five accepted tests are enzyme immunoassay (EIA) for toxin A/B, glutamate dehydrogenase (GDH), nucleic acid amplification tests (NAATs), toxigenic culture (TC), and cytotoxin neutralization (CTN) test. These tests vary widely in terms of clinical usefulness (Table 1)21.\n\n1. NAAT: nucleic acid amplification test; 2. CTN: cytotoxin neutralization test; 3. EIA: solid-phase enzyme immunoassay; 4. glutamate dehydrogenase; 5. toxigenic culture; 6. TAT are variable and dependent on type of NAAT; 7. only available in specialty research laboratories; * indicates magnitude of characteristic, i.e. * has a greater cost than \n\nToxins A and B are the most important virulence determinants of disease and the majority of diagnostic tests target these toxins22. These toxins are responsible for symptoms of infection and are present in the stool of infected patients with diarrhea. The first test for detection, the CTN test, was developed in the 1970s23. CTN was novel in that it detected C. difficile toxins on cell culture medium. Unfortunately, CTN requires significant expertise, is time consuming, has very slow TAT, and is not widely available21,24.\n\nSubsequently, TC on selective medium was developed for the detection of C. difficile23. Although considered the gold standard for its time due to its very high sensitivity, it lacks specificity. Data now show a high rate of false positives in asymptomatic carriers and in certain patient populations, such as infants and patients recently exposed to antibiotics2. In addition, it has a very slow TAT and is not widely available, as testing requires an experienced laboratory21.\n\nIn the early 1990s, detection of C. difficile toxins A and B through solid-phase EIAs was developed. EIAs have a rapid TAT, are widely available and inexpensive, and thus became the new standard for CDI detection in most laboratories until the early 2000s21. Although initially reported to have a sensitivity of as high as 98%, subsequent studies showed toxin A and B EIA had a poorer sensitivity, between 45 and 60%, respectively, but a positive predictive value between 90 and 100%, respectively22. Currently, the general consensus is that the EIA for toxin A and B is too insensitive and is no longer recommended as a stand-alone test11.\n\nIn 2006, the GDH assay was marketed as a CDI detection test. GDH detects C. difficile cell-wall-associated antigen and has a reported sensitivity of 100%. To its strength, GDH has a negative predictive value approaching 100%, but with a positive predictive value of only 59%25. It has a rapid TAT, is widely available and affordable, and has become an effective screening tool for CDI detection21. GDH, however, detects all C. difficile, including nontoxigenic strains, subsequently lowering the specificity for the diagnosis of CDI26.\n\nGiven the high specificity of toxin A/B EIAs and the sensitivity of GDH, several laboratories adopted a two-step algorithm for testing. Referred to as a multistep approach, CDI testing begins with common antigen GDH. If GDH is found to be positive, the toxin A/B assay is performed for the detection of direct toxin production27. GDH and EIA have subsequently been combined and marketed as a single confirmatory test for CDI. The C. Diff QUIK CHEK Complete assay (TechLab, Blacksburg, VA) combines GDH testing and toxin testing using a toxin A/B EIA28. This assay takes about 30 minutes to perform and has a built-in control. At least two publications demonstrate sensitivities of 100% for the GDH portion of the test24,25. The combination of the two tests together in a step-wise process is recognized as confirmation of CDI15,16. Unfortunately, testing can produce discordant results, which can be difficult to interpret, and thus confirmation requires further diagnostic testing20,21.\n\nFurther advancements in detection came in 2009 when NAATs for CDI became commercially available29. The basis of NAATs is the detection of toxigenic C. difficile strains based on DNA extraction from the stool21. In general, the target of most NAATs is the gene responsible for coding toxin B (tcdB gene)29,30. At this time, there are nine US Food and Drug Administration (FDA)-approved C. difficile NAATs. Six are polymerase chain reaction (PCR)-based assays and three are isothermal assays. The assays have sensitivities ranging from 80 to 100%, specificities ranging from 87 to 99%, and all have rapid TATs. NAATs have quickly become popular, and in many laboratories they have become a stand-alone approach for the diagnosis of CDI21,29. NAATs have also been shown to lead to a more rapid diagnosis when compared to GDH and EIA31. There are some data showing earlier detection has led to fewer CDI-related complications, such as intensive care unit admission, colectomy, and death6. However, NAATs have been criticized for being overly sensitive, and their use as a stand-alone test has been controversially linked to elevated reported incidence rates of CDI7,20,29. False positives can occur with NAATs, as they do not detect the presence of biologically active toxin in stool specimens and can detect only the genes responsible for potential toxin production. This has led many to believe that over-diagnosis of colonized C. difficile patients is occurring and that NAATs have increased antibiotic treatment for possible colonized states or limited infections10,20,32.\n\nThe best standard laboratory test for the diagnosis of CDI has not yet been defined; however, recent clinical guidelines on this topic have been published by the Society for Healthcare Epidemiology of America (SHEA), the Infectious Diseases Society of America (IDSA)15, the American College of Gastroenterology (ACG)16, and the United Kingdom National Health Service26. In the United States, the ACG recommends the use of the NAAT as the best test for CDI diagnosis, either as a stand-alone test or as part of a multistep testing algorithm. The ACG also states GDH testing can be used in a two- to three-step algorithm that includes subsequent toxin A/B EIA testing16. The IDSA recommends a two-step method that uses GDH as initial screening followed by the CTN or TC as a confirmatory test. The IDSA recognizes the potential value of NAATs; however, it does not currently recommend these tests in the diagnosis of CDI, citing more data on utility is necessary16. In the United Kingdom, guidelines recommend a combination of two tests, the first of which should be a NAAT or GDH followed by a toxin EIA test33. All guidelines make a significant contribution to clinical decision making, but recent updates should also be considered when choosing testing and treatment.\n\nThere has recently been much research in the field of CDI treatment. New medications and novel therapy highlight the progression made. The first step in treating CDI is to stop the offending antibiotic when possible. Although it is difficult in the age of polypharmacy to accurately quantify the association between antibiotics and CDI, most studies have determined a link between prior exposure to antimicrobial agents and CDI12–14. Medical treatment of CDI varies based on a graded severity scale and whether it is the first occurrence or recurrence of the disease. Severity is usually defined by factors such as age, temperature, serum albumin, and white blood cell count. Guidelines recommend the use of metronidazole 500 mg orally three times per day for 10–14 days for initial mild to moderate disease, and vancomycin 125 mg orally four times per day for 10–14 days for initial severe disease15,16,34. The evidence for these guidelines is supported by Zar et al.’s randomized, prospective, double-blind, placebo-controlled trial, showing vancomycin to be superior to metronidazole in curing severe cases of CDI (97% vs. 76% of patients respectively, p=0.02)35. In patients with mild disease, cure rates were similar in the two treatment groups35. Another study comparing treatment of CDI with vancomycin or metronidazole in patients risk-stratified by infection severity showed significantly less treatment-refractory disease after treatment with vancomycin in severe cases of CDI (32% refractory disease in pre-implementation phase vs. 15% in post-implementation phase, p=0.035)36. Additionally, in another randomized control trial, vancomycin was found to be superior to metronidazole in terms of clinical success and cure rates in patients with severe CDI (88% vs. 77% in the vancomycin and metronidazole groups, respectively)37.\n\nThere is a high risk of recurrence associated with CDI. Studies show that up to 25–30% of patients appropriately treated for CDI experience at least one additional episode15,34. Recurrence comprises both episodes of relapse with infection by the current strain and reinfection by a new strain, and it remains difficult to distinguish between the two infections37,38. Treatment of the first episode of recurrence is usually with the same antibiotic used to treat the initial episode; however, treatment should also be guided by CDI severity if there is a significant change. To help combat the increasing burden of recurrence, the FDA approved fidaxomicin (FDX) for the treatment of CDI in 2011. FDX is a non-absorbed macrolide antibiotic effective against Gram-positive anaerobes but with no effect against bacteroides, a prominent constituent of the intestinal flora. Unlike metronidazole and vancomycin, which both have activity against bacteroides, FDX is thought to have some intestinal microbiota-sparing effect34. FDX has been shown to be superior in the prevention of recurrence of CDI3,39. A large randomized control trial comparing FDX to vancomycin demonstrated a lower rate of recurrence in the FDX group34,39. In another randomized double-blind trial comparing FDX to vancomycin, clinical response rates were similar in the treatment of a first recurrence of CDI; however, FDX was shown to be more likely to prevent a second recurrence34. There are concerns about the cost of FDX, as it is nearly 10-times more expensive than current standard oral vancomycin40. However, a recent study assessed the economic impact of treatment with FDX compared to oral vancomycin and showed an overall cost benefit in patients treated with FDX40. Patients treated with FDX had lower rates of recurrence, lower rates of hospital readmission, and shorter hospital stays, resulting in an overall saving of $3047 per patient treated with FDX40. Although this study has its limitations, it promotes further advancements in the future of CDI treatment with reduced rates of recurrence.\n\nOne existing concept for the treatment of CDI that is gaining popularity is bacteriotherapy with fecal microbiota transplantation (FMT). FMT has been shown to be an effective treatment for recurrent CDI29. Stool from a healthy donor in the form of a liquid suspension has traditionally been transplanted into the patient’s gastrointestinal tract. This can be performed through a variety of routes including nasogastric tube, nasojejunal tube, upper endoscopy, colonoscopy, or enema, with similar success rates41. The rationale for FMT is to restore a healthier intestinal microbiota in patients with recurrent CDI who have disrupted intestinal flora and decreased microbiota diversity from antibiotic therapy42. FMT was previously considered a therapy of last resort for CDI; however, there has been significant research and interest in FMT, and it is becoming more widely practiced41–43. A case series of 12 patients with recurrent CDI treated with FMT demonstrated a 100% cure rate44. Another case series of 18 patients demonstrated a 94% cure rate in the 16 surviving patients45.\n\nA randomized control trial in 2013 compared FMT with donor feces solution transmitted via nasoduodenal tube preceded by four doses of vancomycin and bowel lavage vs. standard vancomycin with and without bowel lavage46. This study showed resolution of diarrhea in 81% of patients after the first FMT and in 94% of patients overall, as two patients were subsequently cured after second infusion of donor feces. Comparatively, only 31% of patients in the vancomycin alone group and 23% in the vancomycin with bowel lavage group had resolution of diarrhea46. Adverse events included diarrhea (94%) immediately after donor-feces infusion, as well as cramping (31%), constipation (19%), and belching (19%). No persistent adverse events related to FMT were noted. The most recent and largest systematic review with meta-analysis in 2015 of FMT studies, involving 18 observational studies with 611 patients, showed a primary cure rate of 91.2% (95% confidence interval [CI] 86.7–94.8%). The overall recurrence rate of CDI was 5.5% (95% CI 2.2–10.3%). The early recurrence rate and late recurrence rate were 2.7% (95% CI 0.7–6.0%) and 1.7% (95% CI 0.4–4.2%), respectively. Most adverse events were expected, short-lived, self-limited, and manageable47. These studies seem to show that FMT is a highly effective therapy for recurrent CDI.\n\nAnother advancement has been the use of probiotics to prevent the development of CDI. Since antibiotics disturb the natural intestinal flora, leading to susceptibility to infection from C. difficile, a treatment which prevents alteration of the natural intestinal microbiome is theorized to help prevent CDI48. The use of lactobacillus has been shown to reduce diarrheal symptoms and reduce the risk of CDI in hospitalized patients on antibiotics49. A large meta-analysis (Cochrane review) composed of 23 randomized controlled trials with 4213 patients showed a significant relative risk reduction in the incidence of C. difficile-associated diarrhea in patients treated with probiotics49. In contrast, a large prospective randomized control trial composed of 3981 patients compared the incidence of antibiotic-associated diarrhea, including C. difficile-associated diarrhea, in patients receiving probiotics compared to a placebo group and found similar incidences of antibiotic-associated diarrhea in the probiotic and placebo groups50. Overall, strong evidence to support the use of probiotic use in the treatment or prevention of CDI is lacking. However, given the overall low cost and lack of significant side effects with probiotics, they are often used to attempt prevention of CDI in patients prescribed antibiotics.\n\nIn summary, many recent efforts and advancements have been made in the diagnosis and treatment of CDI. Rapid and accurate detection of CDI has improved significantly, but possibly at the cost of over-diagnosis. There is still no uniform agreement regarding the best means of diagnosing CDI. Also, when discordant results occur with testing, this may lead to confusion regarding therapy. Future treatment of CDI seems promising, as recent advancements in newer antibiotic therapy and FMT have been shown to more effectively treat CDI, especially in terms of lowering rates of recurrence and also in the treatment of recurrent infection. With the rising burden of CDI, continued research in diagnostic testing and treatment is needed to combat this significant health care problem.\n\n\nAbbreviations\n\nCDI: Clostridium difficile infection\n\nCI: confidence interval\n\nCTN: cytotoxin neutralization test\n\nEIA: enzyme immunoassay\n\nFMT: fecal microbiota transplantation\n\nGDH: glutamate dehydrogenase\n\nNAAT: nucleic acid amplification test\n\nTAT: turn around time", "appendix": "Competing interests\n\n\n\nThe authors declare that they 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\nMagill SS, Edwards JR, Bamberg W, et al.: Multistate point-prevalence survey of health care-associated infections. N Engl J Med. 2014; 370(13): 1198–208. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nKyne L, Warny M, Qamar A, et al.: Asymptomatic carriage of Clostridium difficile and serum levels of IgG antibody against toxin A. N Engl J Med. 2000; 342(6): 390–7. PubMed Abstract \| Publisher Full Text\n\nLo Vecchio A, Zacur GM: Clostridium difficile infection: an update on epidemiology, risk factors, and therapeutic options. Curr Opin Gastroenterol. 2012; 28(1): 1–9. PubMed Abstract \| Publisher Full Text\n\nCenters for Disease Control and Prevention (CDC): Vital signs: preventing Clostridium difficile infections. MMWR Morb Mortal Wkly Rep. 2012; 61(9): 157–62. PubMed Abstract\n\nLucado J, Gould C, Elixhauser A: Clostridium difficile Infections (CDI) in Hospital Stays, 2009. HCUP Statistical Brief #124. Agency for Healthcare Research and Quality, Rockville, MD. 2012. Reference Source\n\nDubberke ER, Olsen MA: Burden of Clostridium difficile on the healthcare system. Clin Infect Dis. 2012; 55(Suppl 2): S88–92. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLongtin Y, Trottier S, Brochu G, et al.: Impact of the type of diagnostic assay on Clostridium difficile infection and complication rates in a mandatory reporting program. Clin Infect Dis. 2013; 56(1): 67–73. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nMoehring RW, Lofgren ET, Anderson DJ: Impact of change to molecular testing for Clostridium difficile infection on healthcare facility-associated incidence rates. Infect Control Hosp Epidemiol. 2013; 34(10): 1055–61. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nFong KS, Fatica C, Hall G, et al.: Impact of PCR testing for Clostridium difficile on incident rates and potential on public reporting: is the playing field level? Infect Control Hosp Epidemiol. 2011; 32(9): 932–3. PubMed Abstract \| Publisher Full Text\n\nKoo HL, Van JN, Zhao M, et al.: Real-time polymerase chain reaction detection of asymptomatic Clostridium difficile colonization and rising C. difficile-associated disease rates. Infect Control Hosp Epidemiol. 2014; 35(6): 667–73. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nIv EC, Iii EC, Johnson DA: Clinical update for the diagnosis and treatment of Clostridium difficile infection. World J Gastrointest Pharmacol Ther. 2014; 5(1): 1–26. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHensgens MP, Goorhuis A, Dekkers OM, et al.: Time interval of increased risk for Clostridium difficile infection after exposure to antibiotics. J Antimicrob Chemother. 2012; 67(3): 742–8. PubMed Abstract \| Publisher Full Text\n\nRiley TV: Antibiotic-associated diarrhoea. A costly problem. Pharmacoeconomics. 1996; 10(1): 1–3. PubMed Abstract \| Publisher Full Text\n\nPépin J, Valiquette L, Alary ME, et al.: Clostridium difficile-associated diarrhea in a region of Quebec from 1991 to 2003: a changing pattern of disease severity. CMAJ. 2004; 171(5): 466–72. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCohen SH, Gerding DN, Johnson S, et al.: Clinical practice guidelines for Clostridium difficile infection in adults: 2010 update by the society for healthcare epidemiology of America (SHEA) and the infectious diseases society of America (IDSA). Infect Control Hosp Epidemiol. 2010; 31(5): 431–55. PubMed Abstract \| Publisher Full Text\n\nSurawicz CM, Brandt LI, Binion DG, et al.: Guidelines for diagnosis, treatment, and prevention of Clostridium difficile infections. Am J Gastroenterol. 2013; 108(4): 478–98; quiz 499. PubMed Abstract \| Publisher Full Text\n\nBagdasarian N, Rao K, Malani PN: Diagnosis and treatment of Clostridium difficile in adults: a systematic review. JAMA. 2015; 313(4): 398–408. PubMed Abstract \| Publisher Full Text\n\nBoland GW, Lee MJ, Cats AM, et al.: Antibiotic-induced diarrhea: specificity of abdominal CT for the diagnosis of Clostridium difficile disease. Radiology. 1994; 191(1): 103–6. PubMed Abstract \| Publisher Full Text\n\nBartlett JG: Clinical practice. Antibiotic-associated diarrhea. N Engl J Med. 2002; 346(5): 334–9. PubMed Abstract \| Publisher Full Text\n\nPolage CR, Gyorke CE, Kennedy MA, et al.: Overdiagnosis of Clostridium difficile Infection in the Molecular Test Era. JAMA Intern Med. 2015; 175(11): 1792–801. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nGilligan PH: Optimizing the Laboratory Diagnosis of Clostridium difficile Infection. Clin Lab Med. 2015; 35(2): 299–312. PubMed Abstract \| Publisher Full Text\n\nLyerly DM, Krivan HC, Wilkins TD: Clostridium difficile: its disease and toxins. Clin Microbiol Rev. 1988; 1(1): 1–18. PubMed Abstract \| Free Full Text\n\nChang TW, Lin PS, Gorbach SL, et al.: Ultrastructural changes of cultured human amnion cells by Clostridiu difficile toxin. Infect Immun. 1979; 23(3): 795–8. PubMed Abstract \| Free Full Text\n\nGeorge WL, Sutter VL, Citron D, et al.: Selective and differential medium for isolation of Clostridium difficile. J Clin Microbiol. 1979; 9(2): 214–9. PubMed Abstract \| Free Full Text\n\nFenner L, Widmer AF, Goy G, et al.: Rapid and reliable diagnostic algorithm for detection of Clostridium difficile. J Clin Microbiol. 2008; 46(1): 328–30. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nTicehurst JR, Aird DZ, Dam LM, et al.: Effective detection of toxigenic Clostridium difficile by a two-step algorithm including tests for antigen and cytotoxin. J Clin Microbiol. 2006; 44(3): 1145–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nQuinn CD, Sefers SE, Babiker W, et al.: C. Diff Quik Chek complete enzyme immunoassay provides a reliable first-line method for detection of Clostridium difficile in stool specimens. J Clin Microbiol. 2010; 48(2): 603–5. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nSwindells J, Brenwald N, Reading N, et al.: Evaluation of diagnostic tests for Clostridium difficile infection. J Clin Microbiol. 2010; 48(2): 606–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSu W, Mercer J, Van Hal SJ, et al.: Clostridium difficile testing: have we got it right? J Clin Microbiol. 2013; 51(1): 377–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBurnham CD, Carroll KC: Diagnosis of Clostridium difficile infection: an ongoing conundrum for clinicians and for clinical laboratories. Clin Microbiol Rev. 2013; 26(3): 604–30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHumphries RM, Uslan DZ, Rubin Z: Performance of Clostridium difficile toxin enzyme immunoassay and nucleic acid amplification tests stratified by patient disease severity. J Clin Microbiol. 2013; 51(3): 869–73. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nPlanche TD, Davies KA, Coen PG, et al.: Differences in outcome according to Clostridium difficile testing method: a prospective multicentre diagnostic validation study of C difficile infection. Lancet Infect Dis. 2013; 13(11): 936–45. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAdvisory Committee on Antimicrobial Resistance and Healthcare Associated Infections: Updated guidance on the diagnosis and reporting of Clostridium difficile. United Kingdom: National Health Service, 2012. Reference Source\n\nCornely OA, Miller MA, Louie TJ, et al.: Treatment of first recurrence of Clostridium difficile infection: fidaxomicin versus vancomycin. Clin Infect Dis. 2012; 55(Suppl 2): S154–61. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nZar FA, Bakkanagari SR, Moorthi KM, et al.: A comparison of vancomycin and metronidazole for the treatment of Clostridium difficile-associated diarrhea, stratified by disease severity. Clin Infect Dis. 2007; 45(3): 302–7. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nJardin CG, Palmer HR, Shah DN, et al.: Assessment of treatment patterns and patient outcomes before vs after implementation of a severity-based Clostridium difficile infection treatment policy. J Hosp Infect. 2013; 85(1): 28–32. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nJohnson S, Louie TJ, Gerding DN, et al.: Vancomycin, metronidazole, or tolevamer for Clostridium difficile infection: results from two multinational, randomized, controlled trials. Clin Infect Dis. 2014; 59(3): 345–54. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nBarbut F, Jones G, Eckert C: Epidemiology and control of Clostridium difficile infections in healthcare settings: an update. Curr Opin Infect Dis. 2011; 24(4): 370–6. PubMed Abstract \| Publisher Full Text\n\nLouie TJ, Miller MA, Mullane KM, et al.: Fidaxomicin versus vancomycin for Clostridium difficile infection. N Engl J Med. 2011; 364(5): 422–31. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nGallagher JC, Reilly JP, Navalkele B, et al.: Clinical and economic benefits of fidaxomicin compared to vancomycin for Clostridium difficile infection. Antimicrob Agents Chemother. 2015; 59(11): 7007–10. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nCammarota G, Ianiro G, Gasbarrini A: Fecal microbiota transplantation for the treatment of Clostridium difficile infection: a systematic review. J Clin Gastroenterol. 2014; 48(8): 693–702. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nKelly CR, Kahn S, Kashyap P, et al.: Update on Fecal Microbiota Transplantation 2015: Indications, Methodologies, Mechanisms, and Outlook. Gastroenterology. 2015; 149(1): 223–37. PubMed Abstract \| Publisher Full Text\n\nDupont HL: Diagnosis and management of Clostridium difficile infection. Clin Gastroenterol Hepatol. 2013; 10(10): 1216–23; quiz e73. PubMed Abstract \| Publisher Full Text\n\nYoon SS, Brandt LI: Treatment of refractory/recurrent C. difficile-associated disease by donated stool transplanted via colonoscopy: a case series of 12 patients. J Clin Gastroenterol. 2010; 44(8): 562–6. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nAas J, Gessert CE, Bakken JS: Recurrent Clostridium difficile colitis: case series involving 18 patients treated with donor stool administered via a nasogastric tube. Clin Infect Dis. 2003; 36(5): 580–5. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nvan Nood E, Vrieze A, Nieuwdorp M, et al.: Duodenal infusion of donor feces for recurrent Clostridium difficile. N Engl J Med. 2013; 368(5): 407–15. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nLi YT, Cai HF, Wang ZH, et al.: Systematic review with meta-analysis: long-term outcomes of faecal microbiota transplantation for Clostridium difficile infection. Aliment Pharmacol Ther. 2016; 43(4): 445–57. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nJohnston BC, Goldenberg JZ, Guyatt GH: Probiotics for the prevention of Clostridium difficile-associated diarrhea. In response. Ann Intern Med. 2013; 158(9): 706–7. PubMed Abstract \| Publisher Full Text\n\nGoldenberg JZ, Ma SS, Saxton JD, et al.: Probiotics for the prevention of Clostridium difficile-associated diarrhea in adults and children. Cochrane Database Syst Rev. 2013; 5: CD006095. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nAllen SJ, Wareham K, Wang D, et al.: Lactobacilli and bifidobacteria in the prevention of antibiotic-associated diarrhoea and Clostridium difficile diarrhoea in older inpatients (PLACIDE): a randomised, double-blind, placebo-controlled, multicentre trial. Lancet. 2013; 382(9900): 1249–57. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation" }	[ { "id": "12195", "date": "29 Jan 2016", "name": "Kevin Garey", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12196", "date": "29 Jan 2016", "name": "Glen Tillotson", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12197", "date": "29 Jan 2016", "name": "Vincent Young", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-118
https://f1000research.com/articles/5-117/v1	29 Jan 16	{ "type": "Review", "title": "Dietary restriction with and without caloric restriction for healthy aging", "authors": [ "Changhan Lee", "Valter Longo" ], "abstract": "Caloric restriction is the most effective and reproducible dietary intervention known to regulate aging and increase the healthy lifespan in various model organisms, ranging from the unicellular yeast to worms, flies, rodents, and primates. However, caloric restriction, which in most cases entails a 20–40% reduction of food consumption relative to normal intake, is a severe intervention that results in both beneficial and detrimental effects. Specific types of chronic, intermittent, or periodic dietary restrictions without chronic caloric restriction have instead the potential to provide a significant healthspan increase while minimizing adverse effects. Improved periodic or targeted dietary restriction regimens that uncouple the challenge of food deprivation from the beneficial effects will allow a safe intervention feasible for a major portion of the population. Here we focus on healthspan interventions that are not chronic or do not require calorie restriction.", "keywords": [ "Caloric restriction", "mechanisms of aging", "Dietary restriction", "aging", "healthspan" ], "content": "Introduction\n\nAging is the leading risk factor for many among the major diseases and a key factor in the overall decline of physical and mental performance. Interventions that slow down the aging process can delay or prevent multiple chronic diseases and improve productivity and quality of life in older ages. For clarity, here, we use caloric restriction (CR) to refer to a dietary intervention with an overall 20–40% reduction of total caloric intake, and dietary restriction to represent a broader scope of dietary interventions that encompass those with specific macronutrient and feeding pattern restrictions. CR is the most studied and reproducible non-genetic intervention known to extend healthspan and/or lifespan in organisms, ranging from unicellular yeast to monkeys. It started with a simple experiment where a reduction in dietary intake (i.e. caloric restriction) extended the lifespan of rats1, providing a foundation to experimentally study the relationship between nutrition and the biology of aging. Here we discuss more recent discoveries that have advanced our knowledge of the effects of less invasive and restrictive dietary interventions in aging and healthspan.\n\n\nCaloric restriction and the conserved mechanisms of aging\n\nIn their seminal report in 1935, Crowell and McCay demonstrated that simply reducing caloric intake without causing malnutrition nearly doubled the lifespan of rats1, providing an experimental model to begin to demonstrate that aging can be slowed down. Nearly half a century later, Walford and Weindruch reported that “adult-initiated” caloric restriction started at 12 months of age not only increased lifespan but also reduced the incidence of spontaneous cancer by more than 50% in rats2,3. Several decades later, the effect of caloric restriction on healthspan and lifespan has been confirmed in model organisms ranging from unicellular yeast to worms, flies, rodents, and primates4, suggesting a highly conserved effect which may involve common genes. Although the molecular mechanisms that mediate the effect of caloric restriction are still being investigated and debated, there is more widespread acceptance of the hypothesis that caloric restriction and lifespan extension involves the down-regulation of insulin and insulin-like signalling (IIS)5, as well as of the amino signalling target of rapamycin (TOR)-S6 kinase pathway6,7, and the glucose signalling Ras-protein kinase A (PKA) pathway6,8,9.\n\nBecause an in depth discussion of the anti- and pro-aging pathways conserved in model organisms has been covered elsewhere, here we will only mention the most relevant ones10–12. In yeast, down-regulation of (a) the amino acid-sensing TOR and the ribosomal protein S6 kinase (S6K) ortholog Sch9 pathway6, and (b) the Ras-AC-PKA pathway13 are key changes mediating part of the effects of caloric restriction on chronological lifespan, the measurement of cellular survival under non-dividing conditions. In contrast, elevated activity of sirtuin (SIR2) has been described as a key change in the extension of replicative lifespan, measured by counting the number of buds generated by an individual mother cell14,15. In worms, the lifespan extension caused by the inactivation of IIS, or by different forms of caloric restriction, requires Forkhead FoxO transcription factor daf-1616. In flies, the IIS pathway is involved in the effects of caloric restriction17 and, although dFoxo is not required for its longevity effect, its activity can affect the response to caloric restriction18. In rodents, growth hormone (GH) and IGF-1 levels are reduced following caloric restriction19, but the link between dietary restriction, GH and aging is still being investigated, with focus on the genes and pathways regulating longevity in the simple organisms described above. The long-lived GH receptor knock-out (GHRKO) mice, which are resistant to GH, do not exhibit further lifespan extension or health benefits by caloric restriction20,21, but the long-lived GH-deficient Ames mice do22, suggesting a complex involvement of the GH/IGF-1 axis and periphery pathways in the response to caloric restriction.\n\nMuch has been learned about caloric restriction and aging from model organisms, but the ultimate question that lingers is the relevance of these models to human lifespan and healthspan. The rhesus monkeys are the closest model organism to humans in which caloric restriction has been experimentally tested in a controlled environment. Two notable studies performed by independent programs, the National Institute on Aging (NIA) Intramural Research Program and the Wisconsin National Primate Research Center (WNPRC), subjected male and female rhesus monkeys to 30% caloric restriction from levels of baseline caloric intake. The NIA reported no improvement in lifespan but observed a positive trend for the delay of age-related diseases (i.e. healthspan)23, whereas WNPRC reported significant improvement in both lifespan and healthspan4,24. The discrepancies are largely attributed to the different dietary composition and heterogenic genetic background4,23, which have been shown to be a significant factor in rodents25,26. This underscores the importance of diet composition and genetic background and their compatibility when applying caloric restriction to humans. Nonetheless, several studies provide evidence supporting beneficial health effects of caloric restriction for humans. A notable NIH-sponsored controlled randomized study on non-obese individuals, CALERIE (Comprehensive Assessment of the Long-term Effects of Reducing Intake of Energy), recently reported that a two year 25% caloric restriction is feasible for humans and provides health benefits, such as reduced inflammatory markers and cardiometabolic risk factors27–29. However, caloric restriction was associated with reduced bone mineral density and exercise was recommended to offset such adverse effects30. Notably, CALERIE was conducted in three independent centers and involved 218 overweight participants, suggesting that caloric restriction can be beneficial even in a very genetically heterogeneous group31. However, considering the results in monkeys, much longer and larger studies will be needed to know what the effects of CR on human healthspan.\n\nAmong the cellular alterations most closely associated with both caloric restriction and longevity mutations is the resistance to multiple stressors, which in most cases includes resistance to oxidative stress. The ability of caloric restriction to prevent the damage caused by exogenous toxins is likely to be associated with the protection, repair and replacement effects that prevent the age-dependent dysfunction caused by endogenous processes and toxic molecules32. An alternative hypothesis suggests that caloric restriction acts as a mild stressor that promotes hormesis, which refers to the beneficial effects resulting from the cellular responses to mild, repeated stress33. Stress resistance should also be considered an important criterion for the successful development of caloric restriction-mimetic dietary and pharmacological interventions.\n\n\nDietary restriction: macromolecular restriction without caloric restriction\n\nThe definition of dietary restriction has been expanded from an alternative description of caloric restriction to also encompass a broader scope of interventions, including short-term starvation, periodic fasting, fasting-mimetic diets, intermittent fasting, normo-caloric diets with planned deficiencies (in particular macronutrients: proteins, carbohydrates, etc.), and time-restricted feeding. Most of these relatively novel interventions are reported to have beneficial effects on overall health and in some cases longevity. Fasting is an extreme dietary intervention describing either a complete lack of food intake or a 60% or higher food restriction. Intermittent fasting refers to practicing this intervention every other day whereas periodic fasting refers to severe restriction for two or more days periodically (every two weeks, month, etc.). Caloric restriction and fasting share similar but often distinct effects on a number of biomarkers (e.g. reduced glucose, and insulin levels) suggesting that partially overlapping mechanisms are involved19. Both intermittent and periodic fasting can increase lifespan, even when there is little or no overall decrease in calorie intake34,50.\n\nIn addition to periodic or intermittent fasting-based strategies as alternatives to caloric restriction, the restriction of specific macronutrients (or macronutrient restriction) without the restriction of calories is among the most promising interventions that have emerged to promote healthy aging in humans. Among the different types of macronutrient restriction, reduced intake of proteins and amino acids is the most effective pro-longevity intervention35,36. Simply reducing protein intake can deliver an equally potent impact on lifespan as dietary restriction in multiple model organisms35. A recent analysis of the National Health and Nutrition Examination Survey (NHANES) showed that low protein intake was associated with reduced overall mortality for those under 65 years of age37. Also, a high-carbohydrate, low-protein diet resulted in longer lifespan and improved cardiometabolic health, despite increased food intake and body fat38,39. Furthermore, the restriction of a single essential amino acid in a normal diet increased lifespan and stress resistance40–44. In flies, adding back essential amino acids to the caloric restriction diet decreased lifespan to that of the normally fed group36. Laboratory rodents fed a methionine-restricted diet displayed an extended lifespan with decreased age-dependent diseases and increased resistance to oxidative stress, in part due to increased antioxidant capacity44–48. A tryptophan-restricted diet also provided longevity and reduced age-dependent deterioration42,43,49 but has mainly been explored for neurological benefits, due to its role in serotonin synthesis. A fasting-mimicking diet, consisting of very low calorie and protein that leads to similar physiological response to fasting, including reduced levels of glucose and IGF-1 and increased levels of ketone bodies IGFBP-1, enhanced healthspan and rejuvenated the hematopoietic system while promoting adult neurogenesis50. Further studies on carbohydrate and fat restriction are needed to determine their role in dietary restriction.\n\nFeeding schedule has also been shown to have a significant impact on health and survival. In flies, time-restricted feeding (limited to 12 daytime hours every day) had profound effects on neural, peripheral, and cardiovascular physiology and improved sleep, body weight maintenance, and delayed signs of cardiac aging, under unchanged caloric intake and activity53. When mice were given access to food for only 8–9 hours during the active phase of the day, metabolic diseases induced by a high-fat, high-fructose, and high-sucrose diet, were reduced without lowering caloric intake51. The benefits of time-restricted feeding against such obesogenic diets were proportional to the duration of the fasting each day52. Ad lib feeding during the weekend did not interfere with the protective effects of time-restricted feeding52. Notably, the restricted feeding pattern reversed the progression of pre-existing obesity and type II diabetes, suggesting it has the potential to be a clinically relevant and feasible dietary intervention, useful to prevent and treat obesity and metabolic disorders52. Considering that key metabolic factors, such as 5' AMP-activated protein kinase (AMPK), sirtuins, and protein kinase B (AKT), are regulated by an interplay of circadian rhythm and feeding time54,55, dietary schedules should be more carefully studied in the context of dietary restriction.\n\n\nConclusion\n\nDietary interventions that extend healthspan and lifespan have evolved and have become much better characterized since the original caloric restriction experiments performed by McCay in 1935. We now understand that its effects on aging are not simply the result of the reduced amount of calories consumed, but are also determined by diet composition, and can be achieved by periodic interventions which do not require an overall reduction in calorie intake and which can be achieved without a complete lack of food intake during the periodic fasting cycles. Further studies are important to identify even less invasive and more effective dietary interventions that will cause coordinated and beneficial effects on healthspan.", "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\nMcCay CM, Crowell MF, Maynard LA: The effect of retarded growth upon the length of life span and upon the ultimate body size. 1935. Nutrition. 1989; 5(3): 155–71; discussion 172. 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PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation" }	[ { "id": "12172", "date": "29 Jan 2016", "name": "Leanne Redman", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12173", "date": "29 Jan 2016", "name": "Julie Mattison", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12174", "date": "29 Jan 2016", "name": "Rozalyn Anderson", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-117
https://f1000research.com/articles/4-1188/v1	30 Oct 15	{ "type": "Research Article", "title": "Feeling the future: A meta-analysis of 90 experiments on the anomalous anticipation of random future events", "authors": [ "Daryl Bem", "Patrizio E. Tressoldi", "Thomas Rabeyron", "Michael Duggan", "Patrizio E. Tressoldi", "Thomas Rabeyron", "Michael Duggan" ], "abstract": "In 2011, one of the authors (DJB) published a report of nine experiments in the Journal of Personality and Social Psychology purporting to demonstrate that an individual’s cognitive and affective responses can be influenced by randomly selected stimulus events that do not occur until after his or her responses have already been made and recorded, a generalized variant of the phenomenon traditionally denoted by the term precognition. To encourage replications, all materials needed to conduct them were made available on request. We here report a meta-analysis of 90 experiments from 33 laboratories in 14 countries which yielded an overall effect greater than 6 sigma, z = 6.40, p = 1.2 × 10-10 with an effect size (Hedges’ g) of 0.09. A Bayesian analysis yielded a Bayes Factor of 1.4 × 109, greatly exceeding the criterion value of 100 for “decisive evidence” in support of the experimental hypothesis. When DJB’s original experiments are excluded, the combined effect size for replications by independent investigators is 0.06, z = 4.16, p = 1.1 × 10-5, and the BF value is 3,853, again exceeding the criterion for “decisive evidence.” The number of potentially unretrieved experiments required to reduce the overall effect size of the complete database to a trivial value of 0.01 is 544, and seven of eight additional statistical tests support the conclusion that the database is not significantly compromised by either selection bias or by “p-hacking”—the selective suppression of findings or analyses that failed to yield statistical significance. P-curve analysis, a recently introduced statistical technique, estimates the true effect size of our database to be 0.20, virtually identical to the effect size of DJB’s original experiments (0.22) and the closely related “presentiment” experiments (0.21). We discuss the controversial status of precognition and other anomalous effects collectively known as psi.", "keywords": [ "precognition", "psi", "ESP", "retrocausation", "retro-priming", "parapsychology" ], "content": "\n\nIn 2011, the Journal of Personality and Social Psychology published an article by one of us (DJB) entitled “Feeling the Future: Experimental Evidence for Anomalous Retroactive Influences on Cognition and Affect” (Bem, 2011). The article reported nine experiments that purported to demonstrate that an individual’s cognitive and affective responses can be influenced by randomly selected stimulus events that do not occur until after his or her responses have already been made and recorded, a generalized variant of the phenomenon traditionally denoted by the term precognition. The controversial nature of these findings prompted the journal’s editors to publish an accompanying editorial justifying their decision to publish the report and expressing their hope and expectation that attempts at replication by other investigators would follow (Judd & Gawronski, 2011).\n\nTo encourage replications from the beginning of his research program in 2000, Bem offered free, comprehensive packages that included detailed instruction manuals for conducting the experiments, computer software for running the experimental sessions, and database programs for collecting and analyzing the data. As of September 2013, two years after the publication of his article, we were able to retrieve 69 attempted replications of his experiments and 11 other experiments that tested for the anomalous anticipation of future events in alternative ways. When Bem’s experiments are included, the complete database comprises 90 experiments from 33 different laboratories located in 14 different countries.\n\nPrecognition is one of several phenomena in which individuals appear to have access to “nonlocal” information, that is, to information that would not normally be available to them through any currently known physical or biological process. These phenomena, collectively referred to as psi, include telepathy, access to another person’s thoughts without the mediation of any known channel of sensory communication; clairvoyance (including a variant called remote viewing), the apparent perception of objects or events that do not provide a stimulus to the known senses; and precognition, the anticipation of future events that could not otherwise be anticipated through any known inferential process.\n\nLaboratory-based tests of precognition have been published for nearly a century. Most of the earlier experiments used forced-choice designs in which participants were explicitly challenged to guess on each trial which one of several potential targets would be randomly selected and displayed in the near future. Typical targets included ESP card symbols, an array of colored light bulbs, the faces of a die, or visual elements in a computer display. When a participant correctly predicted the actual target-to-be, the trial was scored as a hit, and performance was typically expressed as the percentage of hits over a given number of trials.\n\nA meta-analysis of all forced-choice precognition experiments appearing in English language journals between 1935 and 1977 was published by Honorton & Ferrari (1989). Their analysis included 309 experiments conducted by 62 different investigators involving more than 50,000 participants. Honorton and Ferrari reported a small but significant hit rate, Rosenthal effect size z/√n = .02, Stouffer Z = 6.02, p = 1.1 × 10-9. They concluded that this overall result was unlikely to be artifactually inflated by the selective reporting of positive results (the so-called file-drawer effect), calculating that there would have to be 46 unreported studies averaging null results for every reported study in the meta-analysis to reduce the overall significance of the database to chance.\n\nJust as research in cognitive and social psychology has increasingly pursued the study of affective and cognitive processes that are not accessible to conscious awareness or control (e.g., Ferguson & Zayas, 2009), research in psi has followed the same path, moving from explicit forced-choice guessing tasks to experiments using subliminal stimuli and implicit or physiological responses. This trend is exemplified by several “presentiment” experiments, pioneered by Radin (1997) and Bierman (Bierman & Radin, 1997) in which physiological indices of participants’ emotional arousal are continuously monitored as they view a series of pictures on a computer screen. Most of the pictures are emotionally neutral, but on randomly selected trials, a highly arousing erotic or negative image is displayed. As expected, participants show strong physiological arousal when these images appear, but the important “presentiment” finding is that the arousal is observed to occur a few seconds before the picture actually appears on the screen—even before the computer has randomly selected the picture to be displayed.\n\nThe presentiment effect has now been demonstrated using a variety of physiological indices, including electrodermal activity, heart rate, blood volume, pupil dilation, electroencephalographic activity, and fMRI measures of brain activity. A meta-analysis of 26 reports of presentiment experiments published between 1978 and 2010 yielded an average effect size of 0.21, 95% CI = [0.13, 0.29], combined z = 5.30, p = 5.7 × 10-8. The number of unretrieved experiments averaging a null effect that would be required to reduce the effect size to a trivial level was conservatively calculated to be 87 (Mossbridge et al., 2012; see also, Mossbridge et al., 2014). A critique of this meta-analysis has been published by Schwarzkopf (2014) and the authors have responded to that critique (Mossbridge et al., 2015).\n\nBem’s experiments can be viewed as direct descendants of the presentiment experiments. Like them, each of his experiments modified a well-established psychological effect by reversing the usual time-sequence of events so that the participant’s responses were obtained before the putatively causal stimulus events occurred. The hypothesis in each case was that the time-reversed version of the experiment would produce the same result as the standard non-time-reversed experiment. Four well-established psychological effects were modified in this way. (See Bem (2011) for more complete descriptions of the experimental protocols.)\n\n\nPrecognitive approach and avoidance\n\nTwo experiments tested time-reversed versions of one of psychology’s oldest and best known phenomena, the Law of Effect (Thorndike, 1898): An organism is more likely to repeat responses that have been positively reinforced in the past than responses that have not been reinforced. Bem’s time-reversed version of this effect tested whether participants were more likely to make responses that would be reinforced in the near future. On each trial of the first experiment (“Precognitive Detection of Erotic Stimuli”), the participant selected one of two curtains displayed side-by-side on a computer screen. After the participant had made a choice, the computer randomly designated one of the curtains to be the reinforced alternative. If the participant had selected that curtain, it opened to reveal an erotic photograph and the trial was scored as a hit; if the participant had selected the other curtain, a blank gray wall appeared and the trial was scored as a miss. In a second experiment (“Precognitive Avoidance of Negative Stimuli”) a trial was scored as a hit if the participant selected the alternative that avoided the display of a gruesome or unpleasant photograph.\n\n\nRetroactive priming\n\nIn recent years, priming experiments have become a staple of cognitive social psychology (Klauer & Musch, 2003). In a typical affective priming experiment, participants are asked to judge as quickly as they can whether a photograph is pleasant or unpleasant and their response time is measured. Just before the picture appears, a positive or negative word (e.g., beautiful, ugly) is flashed briefly on the screen; this word is called the prime. Individuals typically respond more quickly when the valences of the prime and the photograph are congruent (both are positive or both are negative) than when they are incongruent. In the time-reversed version of the procedure, the randomly-selected prime appeared after rather than before participants judge the affective valence of the photograph.\n\n\nRetroactive habituation\n\nWhen individuals are initially exposed to an emotionally arousing stimulus, they typically have a strong physiological response to it. Upon repeated exposures the arousal diminishes. This habituation process is one possible mechanism behind the so-called “mere exposure” effect in which repeated exposures to a stimulus produce increased liking for it (Bornstein, 1989; Zajonc, 1968). It has been suggested that if a stimulus is initially frightening or unpleasant, repeated exposures will render it less negatively arousing and, hence, it will be better liked after the exposures—the usual mere exposure result—but if the stimulus is initially very positive, the repeated exposures will render it boring or less positively arousing and, hence, it will be less well liked after the exposures (Dijksterhuis & Smith, 2002).\n\nIn two time-reversed habituation experiments, pairs of negative photographs matched for equal likeability or pairs of erotic photographs similarly matched were displayed side by side on the screen and the participant was instructed on each trial to indicate which one he or she liked better. After the preference was recorded, the computer randomly selected one of the two photographs to be the habituation target and flashed it subliminally on the screen several times. The hypothesis was that participants would prefer the habituation target on trials with negative photographs but would prefer the nontarget on trials with erotic photographs.\n\nThe three time-reversed effects described above can be viewed as conceptual replications of the presentiment experiments in that all these experiments assessed affective responses to emotionally arousing stimuli before those stimuli were randomly selected and displayed. Whereas presentiment experiments assess physiological responses, Bem’s experiments assessed behavioral responses. Even the photographs used in the two kinds of experiments were drawn primarily from the same source, the International Affective Picture System (IAPS; Lang & Greenwald, 1993), a set of more than 800 digitized photographs that have been rated for valence and arousal.\n\n\nRetroactive facilitation of recall\n\nA commonplace phenomenon of memory is that practicing or rehearsing a set of verbal items facilitates their subsequent recall. Two of Bem’s time-reversed experiments tested whether rehearsing a set of words makes them easier to recall even if the rehearsal takes place after the recall test is administered. Participants were shown 48 common nouns one at a time on the computer screen. They were then given a (surprise) recall test in which they were asked to type out all the words they could recall, in any order. After the participant completed the recall test, the computer randomly selected half the words to serve as practice words and had participants rehearse them in a series of practice exercises. The hypothesis was that this practice would “reach back in time” to facilitate the recall of these words and, thus, participants would recall more of the to-be-practiced words than the control non-practiced words.\n\nThis protocol is methodologically and conceptually quite different from the three time-reversed protocols described above. In those, participants were required to make quick judgments on each trial with no time to reflect on their decisions. The sequence of events within each trial occurred on a time scale of milliseconds and the putatively causal stimulus appeared immediately after each of the participant’s responses. In terms of Kahneman’s (2011) dual-mode theory of cognition—as described in his book, Thinking, Fast and Slow—these experiments required cognitive processing characteristic of System 1, “Fast Thinking” (also see Evans, 2008, and Evans & Stanovich, 2013).\n\nIn contrast, the retroactive facilitation-of-recall protocol confronted participants with a single extended cognitive task that occurred on a time scale of minutes: Presenting the initial list of words took 2-1/2 minutes; the recall test took up to 5 minutes; and the post-test practice exercises took approximately 7 minutes. This allowed participants time to implement deliberate conscious strategies involving working memory, active rehearsal, and verbal categorization, all cognitive processes characteristic of System 2, “Slow Thinking.”\n\nAcross all his experiments, Bem reported a mean effect size (d) of 0.22, with a Stouffer Z of 6.66, p = 2.68 × 10-11 (Bem et al., 2011).\n\nBem’s experiments have been extensively debated and critiqued. The first published critique appeared in the same issue of the journal as Bem’s original article (Wagenmakers et al., 2011). These authors argued that a Bayesian analysis of Bem’s results did not support his psi-positive conclusions and recommended that all research psychologists abandon frequentist analyses in favor of Bayesian ones. Bem et al. (2011) replied to Wagenmakers et al., criticizing the particular Bayesian analysis they had used and demonstrating that a more reasonable Bayesian analysis yields the same conclusions as Bem’s original frequentist analysis. In a similar critique, Rouder & Morey (2011) also advocated a Bayesian approach, criticizing the analyses of both Bem and Wagenmakers et al. Rather than continuing to debate this issue in the context of Bem’s original experiments, we here analyze the current database with both a frequentist analysis and the specific Bayesian analysis recommended by Rouder and Morey for meta-analyses.\n\nRecently, Judd et al. (2012) have argued that psychologists should start treating stimuli statistically as a random factor the same way we currently treat participants. As they acknowledge, this would constitute a major change in practice for psychologists. To illustrate, they re-analyzed several published datasets from psychological journals, including one of Bem’s retroactive priming results, showing that when stimuli are treated as a random factor the results are statistically weaker than reported in the original articles. They conclude that “As our simulations make clear, in many commonly used designs in social cognitive research, a likely consequence of only treating participants as a random effect is a large inflation of Type I statistical errors, well above the nominal .05 rate (p. 12).”\n\nFrancis (2012) and Schimmack (2012) take a different tack. Instead of arguing that Bem’s results are weaker than he reports, they argue that, on the contrary, his results are actually too good to be true. That is, given the statistical power of Bem’s effects, it is unlikely that eight of his nine experiments would have achieved statistical significance, implying that there is a hidden file-drawer of experiments or failed statistical analyses that Bem failed to report.\n\nIn his own discussion of potential file-drawer issues, Bem (2011) reported that they arose most acutely in his two earliest experiments (on retroactive habituation) because they required extensive pre-experiment pilot testing to select and match pairs of photographs and to adjust the number and timing of the repeated subliminal stimulus exposures. Once these were determined, however, the protocol was “frozen” and the formal experiments begun. Results from the first experiment were used to rematch several of the photographs used for its subsequent replication. In turn, these two initial experiments provided data relevant for setting the experimental procedures and parameters used in all the subsequent experiments.\n\nAs Bem’s explicitly stated in his article, he omitted one exploratory experiment conducted after he had completed the original habituation experiment and its successful replication. It used supraliminal rather than subliminal exposures. He noted that this fundamentally alters the participant’s phenomenology of the experiment, transforming the task into an explicit ESP challenge and thereby undermining the very rationale for using an implicit response measure of psi in the first place. Even that experiment was not left languishing in a file drawer, however, because he had reported and critiqued it at a meeting of the Parapsychological Association (Bem, 2003).\n\nWith regard to unreported data analyses, Bem analyzed and reported each experiment with two to four different analyses, demonstrating in each case that the results and conclusions were robust across different kinds of analyses, different indices of psi performance, and different definitions of outliers. Following standard practice, however, he did not treat stimuli as a random factor in his analyses.\n\nIn his own critique, Francis (2012) remarks that “perhaps the most striking characteristic of [Bem’s] study is that [it meets] the current standards of experimental psychology. The implication is that it is the standards and practices of the field that are not operating properly (p. 155).” Similarly, LeBel & Peters (2011) remark that “...[i]t is precisely because Bem’s report is of objectively high quality that it is diagnostic of potential problems with MRP [Modal Research Practice].... Bem has put empirical psychologists in a difficult position: forced to consider either revising beliefs about the fundamental nature of time and causality or revising beliefs about the soundness of MRP (p. 371).”\n\nLeBel and Peters conclude by recommending that we should put a stronger emphasis on replication. We agree. Rather than continuing to debate Bem’s original experiments, we seek in our meta-analysis to answer the one question that most decisively trumps such disputes: Can independent investigators replicate the original experiments?\n\n\nMethod\n\nThe methodology and reporting of results comply with the Meta-Analysis Reporting Standards (APA, 2008). Additional materials needed to replicate our results independently can be found at http://figshare.com/articles/Meta-analysis_Implicit_Behavioral_Anticipation/903716.\n\nAs noted above, the archival summary publication of Bem’s experiments appeared in 2011, but he had begun his first experiments as early as 2000, and began reporting results soon thereafter at departmental colloquia and annual meetings of the Parapsychological Association (Bem, 2003; Bem, 2005; Bem, 2008). Simultaneously he made materials available to anyone expressing an interest in trying to replicate the experiments. As a result, attempted replications of the experiments began to appear as early as 2001 (as reported in Moulton & Kosslyn, 2011).\n\nNo presentiment experiments are included in our database because, as noted above, a meta-analysis of those has already been published (Mossbridge et al., 2012). We have, however, included 19 attempted replications of Bem’s Retroactive-Facilitation-of Recall experiment that had been previously meta-analyzed by Galak et al. (2012) because 8 additional replication studies of that protocol have been reported since then. (This was the only protocol included in Galak et al.’s. meta-analysis.)\n\nAlthough the individual-difference variable of “stimulus seeking” emerged as a significant correlate of psi performance in several of Bem’s original experiments, we have not analyzed that variable in the present meta-analysis because too few of the replications reported on it—especially those that modified Bem’s original protocol.\n\nCo-authors PT, TR, and MD conducted a search for all potentially relevant replications that became available between the year 2000 and September of 2013. These included unpublished reports as well as peer-reviewed, published articles in mainstream psychological journals; specialized journals; proceedings from conferences; and relevant studies found in Google Scholar, PubMed and PsycInfo. The same set of keywords—Bem, feeling the future, precognition— was used for all searches, and no MESH terms or Boolean operators were used. Using email and academia.edu, they also contacted known psi researchers and mainstream researchers who had expressed an interest in replicating Bem’s experiments. Of the ninety-three experiments retrieved, two were eliminated because they were severely underpowered: the first had only one participant; the second had nine (Snodgrass, 2011). A third experiment, reporting positive results, rested on several post-hoc analyses, and so we deemed it too exploratory to include in the meta-analysis (Garton, 2010). The final database thus comprises 90 experiments.\n\nCo-authors PT and TR independently coded and categorized each study with respect to the following variables: a) type of effect(s) tested; b) number of participants enrolled in the study; c) descriptive or inferential statistics used to calculate measures of effect size; d) whether the replication had been conducted before or after the January, 2011 (Online First) publication of Bem’s original experiments; e) whether or not the experiment had been peer-reviewed; and f) type of replication.\n\nFor this last variable, each experiment was categorized into one of three categories: an exact replication of one of Bem’s experiments (31 experiments), a modified replication (38 experiments), or an independently designed experiment that assessed the ability to anticipate randomly-selected future events in some alternative way (11 experiments). To qualify as an exact replication, the experiment had to use Bem’s software without any procedural modifications other than translating on-screen instructions and stimulus words into a language other than English if needed. The eleven experiments that had not been designed to replicate any of Bem’s experiments included five retroactive-priming experiments and six retroactive-practice experiments.\n\nPercentages of agreement for each of the coding variables ranged from a minimum of 90% for the statistical data to 100% for the classification into one of the three categories of experiments. Discrepancies in coding were resolved by discussion between PT and TR.\n\nAll the main inferential statistics, weighted effect-size point estimations with corresponding 95% Confidence Intervals, and combined z values were calculated using the Comprehensive Meta-Analysis software v.2 by Borenstein et al. (2005). Effect sizes (Hedges’ g) and their standard errors were computed from t test values and sample sizes. (Hedges’ g, is similar to the more familiar d [Cohen, 1988], but pools studies using n - 1 for each sample instead of n. This provides a better estimate for smaller sample sizes.) When t test values were not available, we used the effect sizes reported by the authors or estimated them from the descriptive statistics. When more than one dependent variable was measured, a single effect size was calculated by averaging the effect sizes obtained by the different t values.\n\nHeterogeneity within each set of experiments using a particular protocol (e.g., the set of retroactive priming experiments) was assessed using I2 (Huedo-Medina et al., 2006). It estimates the percent of variance across studies due to differences among the true effect sizes. If all the studies are methodologically identical and the subject samples are very similar, then I2 will be small (< 25%) and a fixed-effect model analysis is justified; otherwise a random-effects model is used (Borenstein et al., 2009).\n\nA fixed-effect model assumes that all the studies using a particular protocol have the same true effect size and that the observed variance of effect sizes across the studies is due entirely to random error within the studies. The random-effects model allows for the possibility that different studies included in the analysis may have different true effect sizes and that the observed variation reflects both within-study and between-study sampling error.\n\nA model comparison Bayesian analysis of an experiment pits a specified experimental hypothesis (H1) against the null hypothesis (H0) by calculating the odds that H1 rather than H0 is true—p(H1)/p(H0)—or the reverse. The analysis assumes that each person comes to the data with a subjective prior value for these odds and then adjusts them on the basis of the data to arrive at his or her posterior odds. A Bayesian analysis can be summarized by a number called the Bayes Factor (BF), which expresses the posterior odds independent of any particular individual’s prior odds. For example, a BF of 3 indicates that the observed data favor the experimental hypothesis over the null hypothesis by a ratio of 3:1. The posterior odds for a particular individual can then be calculated by multiplying his or her prior odds by BF. For example, a mildly psi-skeptical individual might initially assign complementary probabilities of .2 and .8 to H1 and H0, respectively, yielding prior odds of .25. If BF = 3 then the Bayesian formula indicates that this individual’s posterior odds should be .75. If BF were to exceed 4, then the posterior odds p(H1)/p(H0) would exceed 1, implying that this individual now favors the experimental hypothesis over the null.\n\nJeffreys (1998) has suggested the following verbal labels for interpreting BF levels of p(H1)/p(H0):\n\nBF = 1 – 3: Worth no more than a bare mention\n\nBF = 3 – 10: Substantial evidence for H1\n\nBF = 10 – 30: Strong evidence for H1\n\nBF = 30 – 100: Very Strong evidence for H1\n\nBF > 100: Decisive evidence for H1\n\nTo perform a Bayesian analysis, one must also specify a prior probability distribution of effect sizes across a range for both H0 and H1. Specifying the effect size for H0 is simple because it is a single value of 0, but specifying H1 requires specifying a probability distribution across a range of what the effect size might be if H1 were in fact true. This specification can strongly impact the subsequent estimates of BF and, in fact, was the major disputed issue in the debate over Bem’s original experiments (Bem et al., 2011; Rouder & Morey, 2011; Wagenmakers et al., 2011).\n\nFor purposes of meta-analysis, Rouder & Morey (2011) argue that one should use the Jeffrey, Zellner and Siow (JZS) prior probability distribution (see, also, Bayarri & Garcia-Donato, 2007). That distribution is designed to minimize assumptions about the range of effect sizes and, in this sense, constitutes what is known as an “objective” prior (Rouder et al., 2009). Moreover, the resulting BF is independent of the measurement scale of the dependent variable, is always finite for finite data, and is consistent in the sense that as sample size increases, BF grows to infinity if the null is false and shrinks to zero if it is true—a consistency that does not obtain for p values. Researchers can also incorporate their expectations for different experimental contexts by tuning the scale of the prior on effect size (designated as r). Smaller values of r (e.g., 0.1) are appropriate when small effects sizes are expected; larger values of r (e.g., 1.0) are appropriate when large effect sizes are expected. As r increases, BF provides increasing support for the null.\n\nFor these several reasons, we have adopted the JZS prior probability distribution for our Bayesian analysis. For the estimation of Bayes Factors, we used the meta.ttest function of the BayesFactor package (Morey & Rouder, 2014). In the expectation that the effect size will be small, we set r = 0.1. To estimate the overall effect size and τ2, a measure of between-studies variance, we employed the DiMaggio (2013) script, which uses the R2jags package to run the “BUGS” program (Bayesian Analysis Using Gibb’s Sampling). This provides a Monte Carlo Markov Chain simulation approach to parameter estimation using a normally distributed prior with a mean of 0.1 and a wide variance of 105. The program chooses samples using either Gibbs or Metropolis Hasting algorithms. Because this is a simulation-based approach, we repeated many draws or iterations and evaluated whether the chain of sample values converged to a stable distribution, which was assumed to be the posterior distribution in which we are interested.\n\nWe ran two 20,000 Markov Chain Monte Carlo iterations, each starting with different and dispersed initial values for the model. We based our results on the final 20,000 iterations and assessed whether the chain of values had converged to a stable posterior distribution by monitoring and assessing a graph of the chain and by calculating the Brooks Gelman and Rubin statistic, a tool within the CODA package of R programs for this purpose. The results are presented as mean values of the posterior distributions and their 95% credible intervals (CrI).\n\n\nResults and discussion\n\nThe complete database comprises 90 experiments conducted between 2001 and 2013. These originated in 33 different laboratories located in 14 countries and involved 12,406 participants. The full database with corresponding effect sizes, standard errors, and category assignments is presented in Table S1 along with a forest plot of the individual effect sizes and their 95% confidence intervals.\n\nThe first question addressed by the meta-analysis is whether the database provides overall evidence for the anomalous anticipation of random future events. As shown in the first and second rows of Table 1, the answer is yes: The overall effect size (Hedges’ g) is 0.09, combined z = 6.33, p = 1.2 × 10-10. The Bayesian BF value is 5.1 × 109, greatly exceeding the criterion value of 100 that is considered to constitute “decisive evidence” for the experimental hypothesis (Jeffreys, 1998). Moreover, the BF value is robust across a wide range of the scaling factor r, ranging from a high value of 5.1 × 109 when we set r = 0.1 to a low value of 2.0 × 109 when r = 1.0.\n\nNote. In a Bayesian analysis, the analogue to the 95%CI is Crl, “credible intervals of the posterior distributions.” I2 is an estimate of the percent of variance across studies due to differences among the true effect sizes. τ2 is the between-studies variance.\n\na Assuming a null ES of .01 and a variance of .005 (the observed variance, τ2, in the random-effects model), the statistical power of this meta-analysis is 0.95 (Hedges & Pigott, 2001).\n\nb These analyses exclude Bem’s own experiments and the eleven experiments that had not been designed as replications of those experiments.\n\nThe second question is whether independent investigators can successfully replicate Bem’s original experiments. As shown in the third and fourth rows of Table 1, the answer is again yes: When Bem’s experiments are excluded, the combined effect size for attempted replications by other investigators is 0.06, z = 4.16, p = 1.1 × 10-5, and the BF value is 3,853, which again greatly exceeds the criterion value of 100 for “decisive evidence.”\n\nThe fifth and sixth rows of Table 1 show that the mean effect sizes of exact and modified replications are each independently significant and not significantly different from each other (Mean diff = 0.025; 95% CI [-0.04, 0.09]; z = 0.87, ns).\n\nThe seventh and eighth rows show that the mean effect sizes of replications conducted before and after the January, 2011 (online) publication of Bem’s article are each independently significant and not significantly different from each other (Mean diff = 0.042; 95% CI [.02, 0.10]; z = 0.37, ns).\n\nAnd finally, the bottom two rows of Table 1 show that the mean effect sizes of peer reviewed and not-peer-reviewed replications are each independently significant and identical to each other.\n\nTable 2 displays the meta-analysis of the complete database as a function of experiment type and divided post-hoc into fast-thinking and slow-thinking protocols.\n\na Fixed-effect model\n\nAs shown in Table 2, fast-thinking protocols fared better than slow-thinking protocols: Every fast-thinking protocol individually achieved a statistically significant effect, with an overall effect size of 0.11 and a combined z greater than 7 sigma. In contrast, slow-thinking experiments achieved an overall effect size of only 0.03, failing even to achieve a conventional level of statistical significance (p = .16).\n\nOne possible reason for the less successful performance of the slow-thinking experiments is that 12 of the 27 attempted replications of Bem’s retroactive facilitation of recall experiment were modified replications. The 15 exact replications of that protocol yielded an overall effect size of 0.08, but the 12 modified replications yielded a null effect size (-0.00). For example, Galak et al. (2012) used their own software to conduct seven of their 11 modified replications in which 87% of the sessions (2,845 of 3,289 sessions) were conducted online, thereby bypassing the controlled conditions of the laboratory. These unsupervised sessions produced an overall effect size of -0.02. Because experiments in a meta-analysis are weighted by sample size, the huge N of these online experiments substantially lowers the mean effect size of the replications: When the online experiments are removed, the mean ES for this protocol rises to 0.06 [0.00, 0.12]; z = 1.95, p = .05.\n\nNevertheless, we still believe that it is the fast/slow variable itself that is an important determinant of the lower success rate of the slow-thinking experiments. In particular, we suspect that fast-thinking protocols are more likely to produce evidence for psi because they prevent conscious cognitive strategies from interfering with the automatic, unconscious, and implicit nature of psi functioning (Carpenter, 2012). This parallels the finding in conventional psychology that mere exposure effects are most likely to occur when the exposures are subliminal or incidental because the participant is not aware of them and, hence, is not prompted to counter their attitude-inducing effects (Bornstein, 1989).\n\nFinally, Table 2 reveals that the clear winner of our meta-analytic sweepstakes is the precognitive detection of erotic stimuli (row 1), the time-reversed version of psychology’s time-honored Law of Effect. The fourteen experiments using that protocol— conducted in laboratories in four different countries—achieve a larger effect size (0.14), a larger combined z (4.22), and a more statistically significant result (p = 1.2 × 10-5) than any other protocol in the Table. This protocol was also the most reliable: If we exclude the three experiments that were not designed to be replications of Bem’s original protocol, 10 of the 11 replication attempts were successful, achieving effect sizes ranging from 0.12 to 0.52. The one exception was a replication failure conducted by Wagenmakers et al. (2012), which yielded a non-significant effect in the unpredicted direction, ES = -0.02, t(99) = -0.22, ns. These investigators wrote their own version of the software and used a set of erotic photographs that were much less sexually explicit than those used in Bem’s experiment and its exact replications.\n\nThe results of our meta-analysis do not stand alone. As we noted in the introduction, Bem’s experiments can be viewed as conceptual replications of the presentiment experiments in which participants display physiological arousal to erotic and negative photographs a few seconds before the photographs are selected and displayed (Mossbridge et al., 2012). The parallel is particularly close for the two protocols testing the precognitive detection of erotic stimuli and the precognitive avoidance of negative stimuli (Protocols 1 and 2 in Table 2). Together those two protocols achieve a combined effect size of 0.11, z = 4.74, p = 1.07 × 10-6.\n\n\nFile-drawer effects: Selection bias and P-hacking\n\nBecause successful studies are more likely to be published than unsuccessful studies—the file-drawer effect—conclusions that are drawn from meta-analyses of the known studies can be misleading. To help mitigate this problem, the Parapsychological Association adopted the policy in 1976 of explicitly encouraging the submission and publication of psi experiments regardless of their statistical outcomes. Similarly, we put as much effort as we could in locating unpublished attempts to replicate Bem’s experiments by contacting both psi and mainstream researchers who had requested his replication packages or had otherwise expressed an interest in replicating the experiments. As we saw in Table 1, this all appears to have had the desired effect on the current database: Peer-reviewed experiments yielded the same results as experiments that were not peer-reviewed.\n\nThere are also several statistical techniques for assessing the extent to which the absence of unknown studies might be biasing a meta-analysis. We consider nine of them here.\n\n\nFail-safe calculations\n\nOne of the earliest of these techniques was the calculation of a “Fail-Safe N,” the number of unknown studies averaging null results that would nullify the overall significance level of the database if they were to be included in the meta-analysis (Rosenthal, 1979). The argument was that if this number were implausibly large, it would give us greater confidence in the conclusions based on the known studies. The Rosenthal Fail-Safe N, however, has been criticized as insufficiently conservative because it does not take into account the likely possibility that unpublished or unretrieved studies might well have a mean non-zero effect in the unpredicted direction. Thus the estimate of the Fail-Safe N is likely to be too high. (For the record, the Rosenthal Fail-Safe N for our database is greater than 1,000.)\n\nAn alternative approach for estimating a Fail-Safe N focuses on the effect size rather than the p value (Orwin, 1983). The investigator first specifies two numbers: The first is an average effect size for missing studies which, if added to the database, would bring the combined effect size under a specified “trivial” threshold—the second number that must be specified. If we set the mean effect size of missing studies at .001 and define the threshold for a “trivial” effect size to be .01, then the Orwin Fail-Safe N for our database is 544 studies. That is, there would have to be 544 studies missing from our database with a mean effect size of .001 to reduce its overall effect size to .01.\n\n\nCorrelations between study size and effect size\n\nAnother set of indices for assessing selection bias are various correlational measures for assessing the relationship between the size of a study and its effect size. The most direct is the Begg and Mazumdar’s rank correlation test, which simply calculates the rank correlation (Kendall’s tau) between the variances or standard errors of the studies and their standardized effect sizes (Rothstein et al., 2005). If this correlation is significantly negative, if small underpowered studies have larger effect sizes than larger studies, then there is reason to suspect the presence of publication or retrieval bias in the database. For our database, Kendall’s tau is actually slightly positive: τ = +0.10; z = 1.40, implying that our database is not seriously biased by a selection bias.\n\nMore recent publications (e.g., Jin et al., 2015; Rücker et al., 2011; Schwarzer et al., 2010; Stanley & Doucouliagos, 2014; Stanley & Doucouliagos, 2015) have urged the adoption of more complex indices of selection bias:\n\n1. The Copas method (Copas, 2013; Schwarzer et al., 2010) is based on two models, the standard random effects model and the selection model, which takes study size into account.\n\n2. The Limit meta-analysis (Schwarzer et al., 2014) is an extended random effects model that takes account of possible small-study effects by allowing the treatment effect to depend on the standard error.\n\n3. The Precision Effect Test (PET, Stanley, 2008; Stanley & Doucouliagos, 2014) is a variant of the classical Egger regression test (Sterne & Egger, 2005), which tests the relationship between study size and effect size.\n\n4. The Weighted Least Squares analysis (Stanley & Doucouliagos, 2015) provides estimates that are comparable to random effects analyses when there is no publication bias and are identical to fixed-effect analyses when there is no heterogeneity, providing superior estimates compared with both conventional fixed and random effects analyses.\n\nTable 3 summarizes the results of applying these four additional tests to our database.\n\nAs Table 3 shows, three of the four tests yield significant effect sizes estimates for our database after being corrected for potential selection bias; the PET analysis is the only test in which the 95% confidence interval includes the zero effect size. As Sterne & Egger (2005) themselves caution, however, this procedure cannot assign a causal mechanism, such as selection bias, to the correlation between study size and effect size, and they urge the use of the more noncommittal term “small-study effect.”\n\n\nTrim and fill\n\nCurrently the most common method for estimating the number of studies with low effect sizes that might be missing from a database is Duval & Tweedie’s (2000) Trim-and-Fill procedure. It is based on a graphic display of the correlation between sample size and effect size called the “funnel” plot, which plots a measure of sample size on the vertical axis as a function of effect sizes on the horizontal axis. The funnel plot for our database is displayed in Figure 1, which uses the reciprocal of the standard error as the measure of sample size.\n\nIf a meta-analysis has captured all the relevant experiments, we would expect the funnel plot to be symmetric: Experiments should be dispersed equally on both sides of the mean effect size. If the funnel plot is asymmetric, with a relatively high number of small experiments falling to the right of the mean effect size and relatively few falling to the left, it signals the possibility that there may be experiments with small or null effects that actually exist but are missing from the database under consideration.\n\nUsing an iterative procedure, the trim-and-fill method begins by trimming experiments from the extreme right end of the plot (i.e., the smallest studies with the largest effect sizes) and then calculating a new mean effect size. It then reinserts the trimmed studies on the right and inserts their imputed “missing” counterparts symmetrically to the left of the new mean effect size. This produces a revised, more symmetric funnel plot centered around the newly revised mean effect size. This process continues until the funnel plot becomes symmetric. At that point, the plot is centered around a final corrected estimate of the effect size and displays the number of imputed “missing” experiments to the left of the unbiased mean effect size.\n\nFigure 1 displays the funnel plot for our complete database after it has been modified by the trim-and-fill procedure. The unfilled diamond under the horizontal axis marks the original observed effect size (0.09, see Table 1) and the black diamond marks the corrected estimate of the effect size: 0.07 [0.04, 0.10]. The unfilled circles identify the 90 actual experiments in the meta-analysis; the black circles identify the imputed missing experiments. As Figure 1 shows, there are only eight potentially missing studies. As noted above, the Orwin Fail-Safe estimate of how many missing experiments with low effect sizes would be required to nullify the overall effect size of the database is 544.\n\n\nP-curve analysis\n\nAll the analyses discussed above presume that selection bias is driven by effect-size considerations, but Simonsohn et al. (2014a); Simonsohn et al. (2014b) have argued that it is actually more likely to be driven by the p = .05 significance level. They have also demonstrated empirically that the trim and fill procedure is inadequate for estimating the true effect size present in the database (2014b). In its place, they and other authors (van Assen et al., 2015) have recently proposed a very different approach called p-curve analysis.\n\nP-curve is the distribution of significant (p < .05) results among the experiments in a meta-analysis. “It capitalizes on the fact that the distribution of significant p values... is a function of the true underlying effect. Researchers armed only with sample sizes and test results of the published findings can correct for publication bias (Simonsohn et al., 2014b, p. 666).” In addition to assessing selection bias, p-curve analysis can also assess the presence of “p-hacking,” questionable practices of selective reporting that illegitimately enable an investigator to claim results that meet the coveted p < .05 threshold (Simonsohn, et al., 2014a; Simonsohn, et al., 2014b).\n\nIn our database, 17 (19%) of the 90 studies reported results that were statistically significant at the .05 level. The solid blue line in Figure 2 displays the p-curve distribution of those studies.\n\nThe dotted horizontal red line (“Null of zero effect”) is the distribution expected if there is no effect in the data. In that case, 5% of the significant p values will be below .05, 4% will be below .04, 3% will be below .03, 2% will be below .02, and 1% will be below .01. Thus there will be as many p values between .04 and .05 as between .00 and .01, and the shape of the p-curve is a uniform, straight horizontal line with 20% of the significant values within each of the 5 intervals on the horizontal-axis. If a genuine non-zero effect exists, however, then p-curve’s expected distribution will be right-skewed:\n\nWe expect to observe more low significant p values (p < .01) than high significant p values (.04 < p < .05) (Simonsohn et al., 2014b, pp. 666–667)... A set of significant findings contains evidential value when we can rule out selective reporting as the sole explanation of those findings. Only right-skewed p-curves... are diagnostic of evidential value. P-curves that are not right-skewed suggest that the set of findings lacks evidential value, and curves that are left-skewed suggest the presence of intense p-hacking (Simonsohn et al., 2014a, p. 535).\n\nTable 4 presents the skewness analysis of our database (Simonsohn et al., 2014b).\n\nAs shown in the first row of Table 4, the right-skew of the p-curve is equivocally significant (p = .048, p = .056). When this is the case, Simonsohn et al. (2014a) propose applying a second test to see if the studies lack evidential value because they are flatter than an underpowered (33%) p-curve—depicted by the dashed green line. As shown in the second row of the Table, the observed p-curve is not flatter than the null at 33% power, so we cannot conclude that the evidential value of the database is inadequate. And finally, the bottom row shows that the p-curve is clearly not left-skewed, implying that the database has not been strongly p-hacked.\n\nBecause the right-skew of the p-curve is equivocally significant, we turned to a more direct p-curve algorithm called p-uniform (Van Assen et al., 2015) that directly tests the degree to which the observed curve differs from the “no-effect” uniform distribution. (If there is a substantial amount of heterogeneity in the meta-analysis, this method should be used as a sensitivity analysis.) The p-uniform test confirms that there is, in fact, a significant effect in our database (p = .005) and that there is no evidence for selection bias (p = .857).\n\nIn sum, eight of the nine statistical tests we have applied to our database support the conclusion that its overall statistical significance has not been compromised by either selection bias or by p-hacking.\n\n\nP-curve and the true effect size\n\nOne of the counterintuitive derivations from p-curve analysis—confirmed by extensive simulations—is that when the distribution of significant p values is right-skewed, the inclusion of studies with nonsignificant p levels (p > .05) in a meta-analysis actually underestimates the true effect size in the database (Simonsohn et al., 2014b). Based on the Simonsohn et al. p-curve analysis, the estimate of the true effect size for our database is 0.20, virtually identical to the mean effect size of Bem’s (2011) original experiments (0.22) and the mean effect size of the presentiment experiments (0.21) (Mossbridge et al., 2012). A comparable calculation cannot be legitimately derived from the p-uniform algorithm because it assumes that the population effect size is fixed rather than heterogeneous (van Assen et al., 2015, p. 4). As shown in Table 1, our population effect size is heterogeneous.\n\n\nThe complementary merits of exact and modified replications\n\nOur meta-analysis reveals that both exact and modified replications of Bem’s experiments achieve significant and comparable success rates (Table 1). This is reassuring because the two kinds of replication have different advantages and disadvantages. When a replication succeeds, it logically implies that every step in the replication “worked.” When a replication fails, it logically implies that at least one or more of the steps in the replication failed—including the possibility that the experimental hypothesis is false—but we do not know which step(s) failed. As a consequence, even when exact replications fail, they are still more informative than modified replications because they dramatically limit the number of potential variables that might have caused the failure.\n\nThere is, of course, no such thing as a truly exact replication. For example, the experimenter’s attitudes and expectations remain uncontrolled even in a procedurally exact replication, and there are now more than 345 experiments demonstrating that experimenter attitudes and expectations can produce belief-confirming results, even in simple maze experiments with rats as subjects (Rosenthal & Rubin, 1978).\n\nExact replications also serve to guard against some of the questionable research practices that can produce false-positive results, such as changing the protocol or experimental parameters as the experiment progresses, selectively reporting comparisons and covariates without correcting for the number examined, and selectively presenting statistical analyses that yielded significant results while omitting other analyses that did not (Simmons et al., 2011). By defining an exact replication in our meta-analysis as one that used Bem’s experimental instructions, software, and stimuli, we ensure that the experimental parameters and data analyses are all specified ahead of time. In other words, an exact replication is a publicly available, pre-specified protocol that provides many of the same safeguards against false-positive results that are provided by the preregistration of planned experiments.\n\nDespite the merits of exact replications, however, they cannot uncover artifacts in the original protocol that may produce false positive results, whereas suitably modified replications can do exactly that by showing that an experiment fails when a suspected artifact is controlled for. Modified replications can also assess the generality of an experimental effect by changing some of the parameters and observing whether or not the original results are replicated. For example, the one failed replication of the erotic stimulus detection experiment (Wagenmakers et al., 2012) had substituted mild, non-explicit erotic photographs for the more explicit photographs used in Bem’s original experiment and its exact replications.\n\nAs we noted in the introduction, Judd et al. (2012) have recently suggested that psychologists should begin to treat stimuli statistically as a random factor the same way we currently treat participants. This would constitute a way of testing the generalizability of results in psychological experiments. This would, however, also represent a major change in current practice in psychology, and none of the experiments in our database treated stimuli as a random factor. Nevertheless, some generality of stimuli used in Bem’s experimental protocols is achieved. In those involving erotic photographs, for example, different stimulus sets are used for men and women and all participants are given the choice of viewing opposite-sex or same-sex erotica. Experiments using words as stimuli (e.g., retroactive priming experiments) were successfully replicated in languages other than English.\n\nThe fact that exact and modified replications of Bem’s experiments produced comparable, statistically significant results thus implies generality across stimuli, protocols, subject samples, and national cultures. Moreover, the different protocols can themselves be viewed as conceptual replications of the overarching hypothesis that individuals are capable of anomalously anticipating random future events.\n\n\nGeneral discussion\n\nAs Bem noted in his original 2011 article, psi is a controversial subject, and most academic psychologists do not believe that psi phenomena are likely to exist. A survey of 1,188 college professors in the United States revealed that psychologists were much more skeptical about psi than respondents in the humanities, the social sciences, or the physical sciences, including physics (Wagner & Monnet, 1979). Although this survey is now several years old, many psi researchers have observed that psychologists continue to be the most psi-skeptical subgroup of academics.\n\nAs Bem further noted, there are, in fact, justifiable reasons for the greater skepticism of psychologists. Although our colleagues in other disciplines would probably agree with the oft-quoted dictum that “extraordinary claims require extraordinary evidence,” we psychologists are more likely to be familiar with the methodological and statistical requirements for sustaining such claims and aware of previous claims that failed either to meet those requirements or to survive the test of successful replication. Even for ordinary claims, our conventional frequentist statistical criteria are conservative: The p = .05 threshold is a constant reminder that it is worse to assert that an effect exists when it does not (the Type I error) than to assert that an effect does not exist when it does (the Type II error). (For a refreshing challenge to this view, see Fiedler et al., 2012).\n\nSecond, research in cognitive and social psychology over the past 40 years has sensitized us psychologists to the errors and biases that plague intuitive attempts to draw valid inferences from the data of everyday experience (e.g. Gilovich, 1991; Kahneman, 2011). This leads us to give virtually no weight to anecdotal or journalistic reports of psi, the main source cited in the survey by our colleagues in other disciplines as evidence for their more favorable beliefs about psi.\n\nOne sobering statistic from the survey was that 34% of psychologists in the sample asserted psi to be impossible, more than twice the percentage of all other respondents (16%). Critics of Bayesian analyses frequently point out the reductio ad absurdum case of the extreme skeptic who declares psi or any other testable phenomenon to be impossible. The Bayesian formula implies that for such a person, no finite amount of data can raise the posterior probability in favor of the experimental hypothesis above 0, thereby conferring illusory legitimacy on the most anti-scientific stance. More realistically, all an extreme skeptic needs to do is to set his or her prior odds in favor of the psi alternative sufficiently low so as to rule out the probative force of any data that could reasonably be proffered.\n\nWhich raises the following question: On purely statistical grounds, are the results of our meta-analysis strong enough to raise the posterior odds of such a skeptic to the point at which the psi hypothesis is actually favored over the null, however slightly?\n\nAn opportunity to calculate an approximate answer to this question emerges from the Bayesian critique of Bem’s original experiments made by Wagenmakers et al. (2011). Although they did not explicitly claim psi to be impossible, they came very close by setting their prior odds at 1020 against the psi hypothesis. As shown in Table 1, the Bayes Factor for our database is approximately 109 in favor of the psi hypothesis, which implies that our meta-analysis should lower their posterior odds against the psi hypothesis to 1011. In other words, our “decisive evidence” falls 11 orders of magnitude short of convincing Wagenmakers et al. to reject the null. (See a related analysis of their prior odds in Bem et al., 2011.) Clearly psi-proponents have their work cut out for them.\n\nBeyond this Bayesian argument, a more general reason that many psychologists may find a meta-analysis insufficiently persuasive is that the methodology of meta-analysis is itself currently under intense re-examination, with new procedural safeguards (e.g. preregistration of all included studies) and statistical procedures (e.g., treating stimuli as a random factor, p-curve analysis) appearing almost monthly in the professional literature. Even though our meta-analysis was conceived and initiated prior to many of these developments, we were able to make use of many of them after the fact, (e.g., p-curve analysis) but not others (e.g., preregistration, stimuli treated as a random factor). We thus hope that other researchers will be motivated to follow up with additional experiments and analyses to confirm, disconfirm, or clarify the nature of our findings.\n\nPerhaps the most reasonable and frequently cited argument for being skeptical about psi is that there is no explanatory theory or proposed mechanism for psi phenomena that is compatible with current physical and biological principles. Indeed, this limitation is implied by the very description of psi as “anomalous,” and it provides an arguably legitimate rationale for imposing the requirement that the evidence for psi be “extraordinary.”\n\nWe would argue, however, that this is still not a legitimate rationale for rejecting proffered evidence a priori. Historically, the discovery and scientific exploration of most phenomena have preceded explanatory theories, often by decades (e.g., the analgesic effect of aspirin; the anti-depressant effect of electroconvulsive therapy) or even centuries (e.g., electricity and magnetism, explored in ancient Greece as early as 600 BC, remained without theoretical explanation until the Nineteenth Century). The incompatibility of psi with our current conceptual model of physical reality may say less about psi than about the conceptual model of physical reality that most non-physicists, including psychologists, still take for granted—but which physicists no longer do.\n\nAs is widely known, the conceptual model of physical reality changed dramatically for physicists during the 20th Century, when quantum theory predicted and experiments confirmed the existence of several phenomena that are themselves incompatible with our everyday Newtonian conception of physical reality. Some psi researchers see sufficiently compelling parallels between certain quantum phenomena (e.g., quantum entanglement) and characteristics of psi to warrant considering them as potential mechanisms for psi phenomena (e.g., Broderick, 2007; Radin, 2006). Moreover, specific mechanisms have been proposed that seek to explain psi effects with theories more testable and falsifiable than simple metaphor (e.g., Bierman, 2010; Maier & Buechner, 2015; Walach et al., 2014). A recent collection of these theories is presented in May & Marwaha (2015).\n\nAlthough very few physicists are likely to be interested in pursuing explanations for psi, the American Association for the Advancement of Science (AAAS) has now sponsored two conferences of physicists and psi researchers specifically organized to discuss the extent to which precognition and retrocausation can be reconciled with current or modified versions of quantum theory. The proceedings have been published by the American Institute of Physics (Sheehan, 2006; Sheehan, 2011). A central starting point for the discussions has been the consensus that the fundamental laws of both classical and quantum physics are time symmetric:\n\nThey formally and equally admit time-forward and time-reversed solutions.... Thus, though we began simply desiring to predict the future from the present, we find that the best models do not require—in fact, do not respect—this asymmetry.... [Accordingly,] it seems untenable to assert that time-reverse causation (retrocausation) cannot occur, even though it temporarily runs counter to the macroscopic arrow of time (Sheehan, 2006, p. vii).\n\nIronically, even if quantum-based theories of psi eventually do mature from metaphor to genuinely predictive models, they are still not likely to provide intuitively satisfying descriptive mechanisms for psi because quantum theory itself fails to provide such mechanisms for physical reality. Physicists have learned to live with that conundrum in several ways. Perhaps the most common is simply to ignore it and attend only to the mathematics and empirical findings of the theory—derisively called the “Shut Up and Calculate” school of quantum physics (Kaiser, 2012).\n\nAs physicist and Nobel Laureate Richard Feynman (1994) advised, “Do not keep saying to yourself... ‘but how can it be like that?’ because you will get...into a blind alley from which nobody has yet escaped. Nobody knows how it can be like that (p. 123).”\n\nMeanwhile the data increasingly compel the conclusion that it really is like that.\n\nPerhaps in the future, we will be able to make the same statement about psi.\n\n\nData availability\n\nF1000Research: Dataset 1. Table S1, 10.5256/f1000research.7177.d105136", "appendix": "Author contributions\n\n\n\nP. Tressoldi initiated this project, performed the meta-analyses, and shared with D. Bem the primary responsibility for writing this article; T. Rabeyron and M. Duggan were primarily responsible for retrieving and classifying the studies and were also active contributors to the writing of the article. All authors have seen and agreed to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe are grateful to Charles DiMaggio for his collaboration in implementing Bayesian parameter estimation, to Robbie van Aert and Marcel van Assen for performing their p-uniform analysis on our database, and to Daniel Lakens for his collaboration in preparing the p-curve analysis and critical examination of effect sizes. These helpful colleagues do not necessarily concur with our conclusions.\n\n\nSupplementary materials\n\nForest plot of effect sizes. Each blue dot identifies the estimated effect size for that experiment with the corresponding 95% confidence interval. The red vertical line marks the overall effect size based on the random-effects model.\n\nClick here to access the data.\n\n\nReferences\n\n(References marked with a single asterisk indicate studies included in the meta-analysis)APA Publication and Communication Board Working Group on Journal Article Reporting Standards. Reporting standards for research in psychology: why do we need them? What might they be? Am Psychol. 2008; 63(9): 839–851. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBarušs I, Rabier V: Failure to replicate retrocausal recall. Psychology of Consiousness: Theory, Research, and Practice. 2014; 1(1): 82–91. Publisher Full Text\n\nBatthyany A: A replication of Bem’s retro-priming study. Personal communication. 2008.\n\nBatthyany A: Retroactive/Precognitive Priming: The role of attention allocation on time-reversed affective processing. 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Personal communication. 2006." }	[ { "id": "11511", "date": "17 Dec 2015", "name": "Ina Vitalevna Vasileva", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 the results of meta-analysis 90 experiments performed to check is whether the anomalous anticipation of random future events exist. These results were obtained in 33 different laboratories located in 14 countries and involved 12.406 participants. Daryl Bem initially constructed and realized 4 types of experiments: 1. Precognitive Approach and Avoidance; 2. Retroactive Priming; 3. Retroactive Habituation; 4. Retroactive Facilitation of Recall. In each type of experiment, an influence of time-reserved effects was checked. An effect-size in researches included is enough to support the hypothesis about anomalous anticipation of random future events.Online experiments revealed less successful comparing with traditional experiments with experimenter. Authors suppose that lesser effect-size in online-experiments is caused by inability to control all experimental conditions. I believe that it is an adequate explanation of differences in effect in online and laboratory experiments.Authors discussed a possible influence of experimenter’s belief in psi. It agrees with some hypothetical ideas of “quantum consciousness”. Unfortunately, for three time-reserved experiments (1. Precognitive Approach and Avoidance; 2. Retroactive Priming; 3. Retroactive Habituation) such characteristics as valence and arousal for erotic and unpleasant photographs (The International Affective Picture System) were not described. We could estimate obtained results differential characteristics of participants (gender, age, social status). Particularly, in our recent research (Grigoriev, Vasilieva, 2015) we have obtained the results concerning relationship between such characteristics as a gender, age, satisfaction of basic needs and specific of affective visual stimuli anticipation (similar to “Precognitive Approach and Avoidance” type of experiment described in article and also using stimuli from IAPS) in cohorts of students (men and women), law-abiding and convicted of violent crimes men.Authors also discussed the differences in effect-size between «fast-thinking» and «slow-thinking» strategies. They suggest that fast/slow variable effects on result of psi. From one side I agree with this experimental fact and conclusion, because «fast-thinking», as authors say, «prevent conscious cognitive strategies», although still suppose that detailed research is necessary to continue in respect to these variables, because participants’ strategies may be caused not only with the speed of operation with stimuli, but also with content of stimuli.This article may be accepted for indexing because of its proper methodological and methodical level. Enough variables that may affect psi were taken in consideration.", "responses": [] }, { "id": "11510", "date": "21 Dec 2015", "name": "Paul Grigoriev", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 “Feeling the future: A meta-analysis of 90 experiments on the anomalous anticipation of random future events” by Daryl Bem, Patrizio Tressoldi, Thomas Rabeyron, Michael Duggan raises the fundamental problem: if “nonlocal” anticipation really exists. In his original research Bem (2011) constructed several experimental designs that could indicate time-reversed cognitive and emotional effects similar to well-known and approved in psychology (approach and avoidance, priming, habituation, facilitation of recall). Bem’s innovation to experimental approach in “psi” research is quite prominent because he started to use several methods based not on so-called force-choice. Thus, implicit drives and motivators started playing a role in results. For example, in experiments on “retroactive approach and avoidance” not the fact of guessing by itself is estimated, but the percentage of pictures with some similar content that may actualize similar emotions and drives in most people. More generally, it is proved that intuition and anticipation are more effective and frequent in situations with lack of time and/or information for decision-making (Vasilieva, 2006). In special computer games with unpredicted results based on usage of random number generators we can see notable effect in guessing, especially in first 20 attempts (Li, 1992) while interest is high enough. In present article, when Bem’s results excluded, the effect size became lesser (combined effect size for attempted replications by other investigators is 0.06, z = 4.16, p = 1.1 × 10-5). I do not think as a majority of sceptics, that Bem could select best results anyhow. For my opinion, it may be because Bem’s participants were the students – mostly young people with higher sex drive according to age and social status. In our recent investigation (Grigoriev, Vasilieva, 2015) we got similar results (to Bem’s) within young men (they guessed erotic photos more frequently), but results were opposite for men of higher age (they “guessed” erotic pictures significantly rarer than expected). Really, Bem based his research, in particular, on thesis that participants should be attracted by erotic pictures and move away from unpleasant pictures. Thus, generally we follow our drives or motivations when select photos like we could see the “images of the future” or some contented hints from the future. If we even accept this fantastic assumption, we should remember than each individual is characterized by own structure of needs and psychological defenses, more over they depend from actual psychological and physiological state. So, relatively small effect size may be caused also by total averaging of participants. If we select participants to be similar in some respect, for example, men in prison or students under severe risk of expelling from university, we get more effect size (Grigoriev, Vasilieva, 2015), because real majority of participants were in similar and specific stress situations. Underlying hypothesis about retroactive influence of future events on psyche and physiological state of individual seems to be reasonable in general, although we know nothing about physical mechanisms of such influence except of some metaphors from quantum physics like “nonlocality” on “entanglement”… Still entangled particles are interconnected not only in space, but also in time… Again, there is a place for strong discussion between different physicists. We do not know actually, if entanglement could be enough lasting and existing at high temperatures, and consist enough amount of information within particles like photons, for example. Nevertheless, an argument that it is not worth to study something, only because nobody knows the mechanism of phenomenon, seems to be improper. At least, on the stage of exploratory researches. Moreover, skeptics’ arguments that anticipation of unpredictable events has no any sense for an organism is unqualified. Theory of functional systems of Pyotr Anokhin (Anokhin, 1973) is one of keystones of biology, like the meaning of anticipation (Lomov and Surkov, 1980; Lomov, 1984) for psychological adaptation. We should care only about reproducibility. Yes, there are some difficult problems with reproducibility in investigations of psi in general. Although I do not think that retroactive influence of future events is “psi” at all. We do not know delicate physical and psychological mechanism that may work and provide quite small, but significant effect-size. One could offer at least several ways to overcome such weak reproducibility: 1. We should use longitude (during many days and repeats) for every participant and take into account different variables like physiological state and outer influences like weather, social events etc. 2. Parallel registration of processes in nervous system (McCraty, 2004; Mossbridge et al, 2012; Bierman & Scholte, 2002) to distinguish specific patterns in physiological processes when anomalous perception of unpredictable future events is successful. 3. Higher motivation on success or failure avoidance in participants. 4. Taking into account specific needs and state of every participant that may cause differences of unconscious setting to get the specific results. Of course, this research is still quite odd for traditional psychology and may need some extraordinary evidences. At least authors provide us by enough statistical evidences of effects. The sources of possible artifacts were reviewed on the stages of data collecting, experimental methods and statistical analysis. It is worth to mention, that both Bayesian analysis and effect-size with statistical significance indices are enough high even after elimination of all “suspicious” data. For my opinion, article may be published, because obtained positive results about possibility of anomalous retroactive influence reflect some unknown nonlocal mechanism that acts through time and can be felt by psyche and/or organism. It sounds weird; but we should remember every revolution in science had started from such odd facts, that incorporated then into the new theory… And, as for the problem of weak reproducibility of results, the main strategy here should be the searching for the highly reproducible factors that cause variability of phenomenon. When we discover the reasons of nonreproducibility – thus we could understand a structure of phenomenon better.", "responses": [] } ]	1	https://f1000research.com/articles/4-1188
https://f1000research.com/articles/3-62/v1	19 Feb 14	{ "type": "Commentary", "title": "Software Carpentry: lessons learned", "authors": [ "Greg Wilson" ], "abstract": "Over the last 15 years, Software Carpentry has evolved from a week-long training course at the US National Laboratories into a worldwide volunteer effort to raise standards in scientific computing. This article explains what we have learned along the way, the challenges we now face, and our plans for the future.", "keywords": [ "In January 2012", "John Cook posted this to his widely-read blog1:" ], "content": "Introduction\n\nIn January 2012, John Cook posted this to his widely-read blog1:\n\nIn a review of linear programming solvers from 1987 to 2002, Bob Bixby says that solvers benefited as much from algorithm improvements as from Moore’s law: “Three orders of magnitude in machine speed and three orders of magnitude in algorithmic speed add up to six orders of magnitude in solving power. A model that might have taken a year to solve 10 years ago can now solve in less than 30 seconds”.\n\nA million-fold speed-up is impressive, but hardware and algorithms are only two sides of the iron triangle of programming. The third is programming itself, and while improvements to languages, tools, and practices have undoubtedly made software developers more productive since 1987, the speed-up is percentages rather than orders of magnitude. Setting aside the minority who do high-performance computing (HPC), the time it takes the “desktop majority” of scientists to produce a new computational result is increasingly dominated by how long it takes to write, test, debug, install, and maintain software.\n\nThe problem is that most scientists are never taught how to do this. While their undergraduate programs may include a generic introduction to programming or a statistics or numerical methods course (in which they are often expected to pick up programming on their own), they are almost never told that version control exists, and rarely if ever shown how to design a maintainable program in a systematic way, or how to turn the last twenty commands they typed into a re-usable script. As a result, they routinely spend hours doing things that could be done in minutes, or don’t do things at all because they don’t know where to start2,3.\n\nThis is where Software Carpentry comes in. We ran 91 workshops for over 3500 scientists in 2013. In them, more than 100 volunteer instructors helped attendees learn about program design, task automation, version control, testing, and other unglamorous but time-tested skills4. Two independent assessments in 2012 showed that attendees are actually learning, and applying at least some of what we taught5:\n\nThe program increases participants’ computational understanding, as measured by more than a two-fold (130%) improvement in test scores after the workshop. The program also enhances their habits and routines, and leads them to adopt tools and techniques that are considered standard practice in the software industry. As a result, participants express extremely high levels of satisfaction with their involvement in Software Carpentry (85% learned what they hoped to learn; 95% would recommend the workshop to others).\n\nDespite these generally positive results, many researchers still find it hard to apply what we teach to their own work, and several of our experiments, most notably our attempts to teach online, have been failures.\n\n\nFrom red to green\n\nSome historical context will help explain where and why we have succeeded and failed.\n\nIn 1995–96, the author organized a series of articles in IEEE Computational Science & Engineering titled, “What Should Computer Scientists Teach to Physical Scientists and Engineers?”6. The articles grew out of the frustration he had working with scientists who wanted to run before they could walk, i.e., to parallelize complex programs that were not broken down into self-contained functions, that did not have any automated tests, and that were not under version control7.\n\nIn response, John Reynders (then director of the Advanced Computing Laboratory at Los Alamos National Laboratory) invited the author and Brent Gorda (now at Intel) to teach a week-long course on these topics to LANL staff. The course ran for the first time in July 1998, and was repeated nine times over the next four years. It eventually wound down as the principals moved on to other projects, but two valuable lessons were learned:\n\n1. Intensive week-long courses are easy to schedule (particularly if instructors are travelling) but by the last two days, attendees’ brains are full and learning drops off significantly.\n\n2. Textbook software engineering is not the right thing to teach most scientists. In particular, careful documentation of requirements and lots of up-front design are not appropriate for people who (almost by definition) do not yet know what they are trying to do. Agile development methods, which rose to prominence during this period, are a less bad fit to researchers’ needs, but even they are not well suited to the “solo grad student” model of working so common in science.\n\nThe Software Carpentry course materials were updated and released in 2004–05 under a Creative Commons license thanks to support from the Python Software Foundation8. They were used twice in a conventional term-long graduate course at the University of Toronto aimed at a mix of students from Computer Science and the physical and life sciences.\n\nThe materials attracted 1000–2000 unique visitors a month, with occasional spikes correlated to courses and mentions in other sites. But while grad students (and the occasional faculty member) found the course at Toronto useful, it never found an institutional home. Most Computer Science faculty believe this basic material is too easy to deserve a graduate credit (even though a significant minority of their students, particularly those coming from non-CS backgrounds, have no more experience of practical software development than the average physicist). However, other departments believe that courses like this ought to be offered by Computer Science, in the same way that Mathematics and Statistics departments routinely offer service courses. In the absence of an institutional mechanism to offer credit courses at some inter-departmental level, this course, like many other interdisciplinary courses, fell between two stools.\n\nIt works too well to be interesting\n\nWe have also found that what we teach simply isn’t interesting to most computer scientists. They are interested in doing research to advance our understanding of the science of computing; things like command-line history, tab completion, and “select * from table” have been around too long, and work too well, to be publishable any longer. As long as universities reward research first, and supply teaching last, it is simply not in most computer scientists own best interests to offer this kind of course.\n\nSecondly, despite repeated invitations, other people did not contribute updates or new material beyond an occasional bug report. Piecemeal improvement may be normal in open source development, but Wikipedia aside, it is still rare in other fields. In particular, people often use one another’s slide decks as starting points for their own courses, but rarely offer their changes back to the original author in order to improve them. This is partly because educators’ preferred file formats (Word, PowerPoint, and PDF) can’t be handled gracefully by existing version control systems, but more importantly, there simply isn’t a “culture of contribution” in education for projects like Software Carpentry to build on.\n\nThe most important lesson learned in this period was that while many faculty in science, engineering, and medicine agree that their students should learn more about computing, they won’t agree on what to take out of the current curriculum to make room for it. A typical undergraduate science degree in the US or Canada has roughly 1800 hours of class and laboratory time; anyone who wants to add more programming, statistics, writing, or anything else must either lengthen the program (which is financially and institutionally infeasible) or take something out. However, everything in the program is there because it has a passionate defender who thinks it’s vitally important, and who is likely senior to those faculty advocating the change.\n\nIt adds up\n\nSaying, “We’ll just add a little computing to every other course,” is a cheat: five minutes per hour equals four entire courses in a four-year program, which is unlikely to ever be implemented. Pushing computing down to the high school level is also a non-starter, since that curriculum is also full.\n\nThe sweet spot for this kind of training is therefore the first two or three years of graduate school. At that point, students have time (at least, more time than they’ll have once they’re faculty) and real problems of their own that they want to solve.\n\nThe author rebooted Software Carpentry in May 2010 with support from Indiana University, Michigan State University, Microsoft, MITACS, Queen Mary University of London, Scimatic, SciNet, SHARCNet, and the UK Met Office. More than 120 short video lessons were recorded during the subsequent 12 months, and six more week-long classes were run for the backers. We also offered an online class three times (a MOOC avant la lettre).\n\nThis was our most successful version to date, in part because the scientific landscape itself had changed. Open access publishing, crowd sourcing, and dozens of other innovations had convinced scientists that knowing how to program was now as important to doing science as knowing how to do statistics. Despite this, though, most still regarded it as a tax they had to pay in order to get their science done. Those of us who teach programming may find it interesting in its own right, but as one course participant said, “If I wanted to be a programmer instead of a chemist, I would have chosen computer science as my major instead of chemistry”.\n\nDespite this round’s overall success, there were several disappointments:\n\n1. Once again, we discovered that five eight-hour days are more wearying than enlightening.\n\n2. And once again, only a handful of other people contributed material, not least because creating videos is significantly more challenging than creating slides. Editing or modifying them is harder still: while a typo in a slide can be fixed by opening PowerPoint, making the change, saving, and re-exporting the PDF, inserting new slides into a video and updating the soundtrack seems to take at least half an hour regardless of how small the change is.\n\n3. Most importantly, the MOOC format didn’t work: only 5–10% of those who started with us completed the course, and the majority were people who already knew most of the material. Both figures are in line with completion rates and learner demographics for other MOOCs9, but are no less disappointing because of that.\n\nThe biggest take-away from this round was the need come up with a scalable, sustainable model. One instructor simply can’t reach enough people, and cobbling together funding from half a dozen different sources every twelve to eighteen months is a high-risk approach.\n\nSoftware Carpentry restarted once again in January 2012 with a new grant from the Sloan Foundation, and backing from the Mozilla Foundation. This time, the model was two-day intensive workshops like those pioneered by The Hacker Within, a grassroots group of grad students helping grad students at the University of Wisconsin - Madison.\n\nShortening the workshops made it possible for more people to attend, and increased the proportion of the material they retained. It also forced us to think much harder about what skills scientists really needed. Out went object-oriented programming, XML, Make, GUI construction, design patterns, and software development lifecycles. Instead, we focused on a handful of tools (discussed in the next section) that let us introduce higher-level concepts without learners really noticing.\n\nReaching more people also allowed us to recruit more instructors from workshop participants, which was essential for scaling. Switching to a “host site covers costs” model was equally important: funding is still needed for the coordinator positions (the author and two part-time administrative assistants at Mozilla, and part of one staff member’s time at the Software Sustainability Institute in the UK), but our other costs now take care of themselves.\n\nOur two-day workshops have been an unqualified success. Both the number of workshops, and the number of people attending, have grown steadily (Figure 1 and Figure 2).\n\nMore importantly, feedback from participants is strongly positive. While there are continuing problems with software set-up and the speed of instruction (discussed below), 80–90% of attendees typically report that they were glad they attended and would recommend the workshops to colleagues.\n\n\nWhat we do\n\nSo what does a typical workshop look like?\n\nDay 1 a.m.: The Unix shell. We only show participants a dozen basic commands; the real aim is to introduce them to the idea of combining single-purpose tools (via pipes and filters) to achieve desired effects, and to getting the computer to repeat things (via command completion, history, and loops) so that people don’t have to.\n\nDay 1 p.m.: Programming in Python (or sometimes R). The real goal is to show them when, why, and how to grow programs step-by-step as a set of comprehensible, reusable, and testable functions.\n\nDay 2 a.m.: Version control. We begin by emphasizing how this is a better way to back up files than creating directories with names like “final”, “really_final”, “really_final_revised”, and so on, then show them that it’s also a better way to collaborate than FTP or Dropbox.\n\nDay 2 p.m.: Using databases and SQL. The real goal is to show them what structured data actually is (in particular, why atomic values and keys are important) so that they will understand why it’s important to store information this way.\n\nAs the comments on the bullets above suggest, our real aim isn’t to teach Python, Git, or any other specific tool: it’s to teach computational competence. We can’t do this in the abstract: people won’t show up for a hand-waving talk, and even if they do, they won’t understand. If we show them how to solve a specific problem with a specific tool, though, we can then lead into a larger discussion of how scientists ought to develop, use, and curate software.\n\nWe also try to show people how the pieces fit together: how to write a Python script that fits into a Unix pipeline, how to automate unit tests, etc. Doing this gives us a chance to reinforce ideas, and also increases the odds of them being able to apply what they’ve learned once the workshop is over.\n\nOf course, there are a lot of local variations around the template outlined above. Some instructors still use the command-line Python interpreter, but a growing number have adopted the IPython Notebook, which has proven to be an excellent teaching and learning environment.\n\nWe have also now run several workshops using R instead of Python, and expect this number to grow. While some people feel that using R instead of Python is like using feet and pounds instead of the metric system, it is the lingua franca of statistical computing, particularly in the life sciences. A handful of workshops also cover tools such as LaTeX, or domain-specific topics such as audio file processing. We hope to do more of the latter going forward now that we have enough instructors to be able to specialize.\n\nWe aim for no more than 40 people per room at a workshop, so that every learner can receive personal attention when needed. Where possible, we now run two or more rooms side by side, and use a pre-assessment questionnaire as a sorting hat to stream learners by prior experience, which simplifies teaching and improves their experience. We do not shuffle people from one room to another between the first and second day: with the best inter-instructor coordination in the world, doing so would still result in duplication, missed topics, and jokes that make no sense.\n\nOur workshops are often free, but many now charge a small registration fee (typically $20–40), primarily because it reduces the no-show rate from a third to roughly 5%. When this is done, we must be careful not to trip over institutional rules about commercial use of their space: some universities will charge hundreds or thousands of dollars per day for use of their classrooms if any money changes hands. As this is usually several times more than a small registration fee would bring in, we usually choose the higher no-show rate as the lesser evil.\n\nWe have also experimented with refundable deposits, but the administrative overheads were unsustainable. It also does not help get around the rules mentioned in the previous paragraph, since money still appears to be changing hands in the university’s eyes.\n\nCommercial offerings\n\nOur material10,11 is all covered by the Creative Commons Attribution license, so anyone who wants to use it for corporate training can do so without explicit permission from us. We encourage this: it would be great if graduate students could help pay their bills by sharing what they know, in the way that many programmers earn part or all of their living from working on open source software.\n\nWhat does require permission is use of our name and logo, both of which are trademarked. We are happy to give such permission if we have certified the instructor and have a chance to double-check the content, but we do want a chance to check: we have had instances of people calling something “Software Carpentry” when it had nothing to do with what we usually teach. We’ve worked hard to create material that actually helps scientists, and to build some name recognition around it, and we’d like to make sure our name continues to mean something.\n\nAs well as instructors, we rely on local helpers to wander the room and answer questions during practical sessions. These helpers may be alumni of previous workshops who are interested in becoming instructors, grad students who have picked up some or all of this on their own, or members of the local open source community; where possible, we aim to have at least one helper for every eight learners.\n\nWe find workshops go a lot better if people come in groups (e.g., 4–5 people from one lab) or have other pre-existing ties (e.g., the same disciplinary background). They are less inhibited about asking questions, and can support each other (morally and technically) when the time comes to put what they’ve learned into practice after the workshop is over. Group sign-ups also yield much higher turnout from groups that are otherwise often under-represented, such as women and minority students, since they know in advance that they will be in a supportive environment.\n\n\nSmall things add up\n\nAs in chess, success in teaching often comes from the accumulation of seemingly small advantages. Here are a few of the less significant things we do that we believe have contributed to our success.\n\nWe use live coding rather than slides: it’s more convincing, it enables instructors to be more responsive to “what if?” questions, and it facilitates lateral knowledge transfer (i.e., people learn more than we realized we were teaching them by watching us work). This does put more of a burden on instructors than a pre-packaged slide deck, but most find it more fun.\n\nOur grant proposals, mailing lists, feedback from workshops, and everything else that isn’t personally sensitive are out in the open (see10 for links). While we cannot prove it, we believe that the fact that people can see us actively succeeding, failing, and learning earns us some credibility and respect.\n\nThis is an important special case of the previous point. Anyone who wants to use our lessons can take what we have, make changes, and offer those back by sending us a pull request on GitHub. As mentioned earlier, this workflow is still foreign to most educators, but it is allowing us to scale and adapt more quickly and more cheaply than the centralized approaches being taken by many high-profile online education ventures.\n\nWe also make a point of eating our own cooking, e.g., we use GitHub for our web site and to plan workshops. Again, this buys us credibility, and gives instructors a chance to do some hands-on practice with the things they’re going to teach. The (considerable) downside is that it can be quite difficult for newcomers to contribute material; we are therefore working to streamline that process.\n\nLearners tell us that it is important to them to leave the workshop with their own working environment set up. We therefore continue to teach on all three major platforms (Linux, Mac OS X, and Windows), even though it would be simpler to require learners to use just one. We have experimented with virtual machines on learners’ computers to reduce installation problems, but those introduce problems of their own: older or smaller machines simply aren’t fast enough. We have also tried using virtual machines (VMs) in the cloud, but this makes us dependent on university-quality WiFi.\n\nWe often use Etherpad for collaborative note-taking and to share snippets of code and small data files with learners. (If nothing else, it saves us from having to ask students to copy long URLs from the presenter’s screen to their computers.) It is almost always mentioned positively in post-workshop feedback, and several workshop participants have started using it in their own teaching.\n\nWe are still trying to come up with an equally good way to share larger files dynamically as the lessons progress. Version control does not work, both because our learners are new to it (and therefore likely to make mistakes that affect classmates) and because classroom WiFi frequently can’t handle a flurry of multi-megabyte downloads.\n\nGiving each learner two sticky notes of different colors allows instructors to do quick true/false questions as they’re teaching. It also allows real-time feedback during hands-on work: learners can put a green sticky note on their laptop when they have something completed, or a red one when they need help. We also use them as minute cards: before each break, learners take a minute to write one thing they’ve learned on the green sticky note, and one thing they found confusing (or too fast or too slow) on the red. It only takes a couple of minutes to collate these, and allows the instructors to adjust to learners’ interests and speed.\n\nPairing is a good practice in real life, and an even better way to teach: partners can not only help each other out during the practical, but can also clarify each other’s misconceptions when the solution is presented, and discuss common research interests during breaks. To facilitate this, we strongly prefer flat (dinner-style) seating to banked (theater-style) seating; this also makes it easier for helpers to reach learners who need assistance.\n\nWe are constantly trying out new ideas (though not always on purpose). Among our current experiments are:\n\nPartner and adapt: We have built a very fruitful partnership with the Software Sustainability Institute (SSI), which now manages our activities in the UK, and are adapting our general approach to meet particular local needs.\n\nA driver’s license for HPC: As another example of this collaboration, we are developing a “driver’s license” for researchers who wish to use the DiRAC HPC facility. During several rounds of beta testing, we have refined an hour-long exam to assess people’s proficiency with the Unix shell, testing, Makefiles, and other skills. This exam was deployed in late 2013, and we hope to be able to report on it by mid-2014.\n\nNew channels: On June 24–25, 2013, we ran our first workshop for women in science, engineering, and medicine. This event attracted 120 learners, 9 instructors, a dozen helpers, and direct sponsorship from several companies, universities, and non-profit organizations. Our second such workshop will run in March 2014, and we are exploring ways to reach other groups that are underrepresented in computing.\n\nSmuggling it into the curriculum: Many of our instructors also teach regular university courses, and several of them are now using part or all of our material as the first few lectures in them. We strongly encourage this, and would welcome a chance to work with anyone who wishes to explore this themselves.\n\n\nInstructor training\n\nTo help people teach, we now run an online training course for would-be instructors12. It takes 2–4 hours/week of their time for 12–14 weeks (depending on scheduling interruptions), and introduces them to the basics of educational psychology, instructional design, and how these things apply to teaching programming. It is necessarily very shallow, but most participants report that they find the material interesting as well as useful.\n\nWhy do people volunteer as instructors?\n\nTo make the world a better place. The two things we need to get through the next hundred years are more science and more courage; by helping scientists do more in less time, we are helping with the former.\n\nTo make their own lives better. Our instructors are often asked by their colleagues to help with computing problems. The more those colleagues know, the more interesting those requests are.\n\nTo build a reputation. Showing up to run a workshop is a great way for people to introduce themselves to colleagues, and to make contact with potential collaborators. This is probably the most important reason from Software Carpentry’s point of view, since it’s what makes our model sustainable.\n\nTo practice teaching. This is also important to people contemplating academic careers.\n\nTo help diversify the pipeline. Computing is 12–15% female, and that figure has been dropping since its high point in the 1980s13. Some of our instructors are involved in part because they want to help break that cycle by participating in activities like our workshops for women in science and engineering.\n\nTo learn new things, or learn old things in more detail. Working alongside an instructor with more experience is a great way to learn more about the tools, as well as about teaching.\n\nIt’s fun. Our instructors get to work with smart people who actually want to be in the room, and don’t have to mark anything afterwards. It’s a refreshing change from teaching undergraduate calculus. . .\n\n\nTODO\n\nWe’ve learned a lot, and we’re doing a much better job of reaching and teaching people than we did eighteen months ago, but there are still many things we need to improve.\n\nThe biggest challenge we face is the diversity of our learners’ backgrounds and skill levels. No matter what we teach, and how fast or how slow we go, 20% or more of the room will be lost, and there’s a good chance that a different 20% will be bored.\n\nThe obvious solution is to split people by level, but if we ask them how much they know about particular things, they regularly under- or over-estimate their knowledge. We have therefore developed a short pre-assessment questionnaire (listed in the Supplementary materials) that asks them whether they could accomplish specific tasks. While far from perfect, it seems to work well enough for our purposes.\n\nOur second-biggest problem is financial sustainability. The “host site covers costs” model allows us to offer more workshops, but does not cover the two full-time equivalent coordinating positions at the center of it all. We do ask host sites to donate toward these costs, but are still looking for a long-term solution.\n\nThird, while we believe we’re helping scientists, we have not yet done the long-term follow-up needed to prove this. This is partly because of a lack of resources, but it is also a genuinely hard problem: no one knows how to measure the productivity of programmers, or the productivity of scientists, and putting the two together doesn’t make the unknowns cancel out.\n\nWhat we’ve done so far is collect verbal feedback at the end of every workshop (mostly by asking attendees what went well and what didn’t) and to administer surveys immediately before and afterwards. Neither has been done systematically, though, which limits the insight we can actually glean. We are taking steps to address this, but the larger question of what impact we’re having on scientists’ productivity still needs to be addressed.\n\nMeeting our own standards\n\nOne of the reasons we need to do long-term follow-up is to find out for our own benefit whether we’re teaching the right things the right way. As just one example, some of us believe that Subversion is significantly easier for novices to understand than Git because there are fewer places data can reside and fewer steps in its normal workflow. Others believe just as strongly that there is no difference, or that Git is actually easier to learn. While the large social network centered around GitHub is a factor in our choice as well, we would obviously be able to make better decisions if we had more quantitative data to base them on.\n\nFourth, getting software installed is often harder than using it. This is a hard enough problem for experienced users, but almost by definition our audience is inexperienced, and our learners don’t (yet) know about system paths, environment variables, the half-dozen places configuration files can lurk on a modern system, and so on. Combine that with two versions of Mac OS X, three of Windows, and two oddball Linux distributions, and it’s almost inevitable that every time we introduce a new tool, it won’t work as expected (or at all) for at least one person in the room. Detailed documentation has not proven effective: some learners won’t read it (despite repeated prompting), and no matter how detailed it is, it will be incomprehensible to some, and lacking for others.\n\nEdit this\n\nAnd while it may seem like a trivial thing, editing text is always harder than we expect. We don’t want to encourage people to use naive editors like Notepad, and the two most popular legacy editors on Unix (Vi and Emacs) are both usability nightmares. We now recommend a handful of GUI editors, but it remains a stumbling block.\n\nChallenge number five is to move more of our teaching and follow-up online. We have tried several approaches, from MOOC-style online-only offerings to webcast tutorials and one-to-one online office hours via internet phone calls and desktop sharing. In all cases, turnout has been mediocre at the start and dropped off rapidly. The fact that this is also true of most high-profile MOOCs is little comfort.\n\nSixth on our list is the tension between teaching the “what” and the “how” of programming. When we teach a scripting language like Python, we have to spend time up front on syntax, which leaves us only limited time for the development practices that we really want to focus on, but which are hard to grasp in the abstract. By comparison, version control and databases are straightforward: what you see is what you do is what you get.\n\nWe also don’t as good a job as we would like teaching testing. The mechanics of unit testing with an xUnit-style framework are straightforward, and it’s easy to come up with representative test cases for things like reformatting data files, but what should we tell scientists about testing the numerical parts of their applications? Once we’ve covered floating-point roundoff and the need to use “almost equal” instead of “exactly equal”, our learners quite reasonably ask, “What should I use as a tolerance for my computation?” for which nobody has a good answer.\n\nWhat we actually teach varies more widely than the content of most university courses with prescribed curricula. We think this is a strength, and one of the reasons we recruit instructors from among scientists is that they are best placed to customize content and delivery for local needs. However, we do need to be more systematic about varying our content on purpose rather than by accident.\n\nFinally, we try to make our teaching as interactive as possible, but we still don’t give learners hands-on exercises as frequently as we should. We also don’t give them as diverse a range of exercises as we should, and those that we do give are often at the wrong level. This is partly due to a lack of time, but disorganization is also a factor.\n\nThere is also a constant tension between having students do realistic exercises drawn from actual scientific workflows, and giving them tasks that are small and decoupled, so that failures are less likely and don’t have knockon effects when they occur. This is exacerbated by the diversity of learners in the typical workshop, though we hope that will diminish as we organize and recruit along disciplinary lines instead of geographically.\n\nComputing education researchers have learned a lot in the past two decades about why people find it hard to learn how to program, and how to teach them more effectively14–18. We do our best to cover these ideas in our instructor training program, but are less good about actually applying them in our workshops.\n\n\nConclusions\n\nTo paraphrase William Gibson, the future is already here: it’s just that the skills needed to implement it aren’t evenly distributed. A small number of scientists can easily build an application that scours the web for recently-published data, launch a cloud computing node to compare it to home-grown data sets, and push the result to a GitHub account; others are still struggling to free their data from Excel and figure out which of the nine backup versions of their paper is the one they sent for publication.\n\nThe fact is, it’s hard for scientists to do the cool things their colleagues are excited about without basic computing skills, and impossible for them to know what other new things are possible. Our ambition is to change that: not just to make scientists more productive today, but to allow them to be part of the changes that are transforming science in front of our eyes. If you would like to help, we’d like to hear from you: please mail us at admin@software-carpentry.org.\n\n\nData availability\n\nfigshare: Attendance and number of Software Carpentry workshops January 2012–January 2014, doi: 10.6084/m9.figshare.92854719", "appendix": "Competing interests\n\n\n\nThe author is an employee of the Mozilla Foundation. Over the years, Software Carpentry has received support from: The Sloan Foundation, Microsoft, NumFOCUS, Continuum Analytics, Enthought, The Python Software Foundation, Indiana University, Michigan State University, MITACS, The Mozilla Foundation, Queen Mary University London, Scimatic Inc., SciNET, SHARCNET, The UK Met Office, The MathWorks, Los Alamos National Laboratory, Lawrence Berkeley National Laboratory.\n\n\nGrant information\n\nSoftware Carpentry is currently supported by a grant from the Sloan Foundation.\n\n\nAcknowledgements\n\nThe author wishes to thank Brent Gorda, who helped create Software Carpentry sixteen years ago; the hundreds of people who have helped organize and teach workshops over the years; and the thousands of people who have taken a few days to learn how to get more science done in less time, with less pain. Particular thanks go to the following for their comments, corrections, and inspiration:\n\nAzalee Bostroem (Space Telescope Science Institute)\n\nChris Cannam (Queen Mary, University of London)\n\nStephen Crouch (Software Sustainability Institute)\n\nMatt Davis (Datapad, Inc.)\n\nLuis Figueira (King’s College London)\n\nRichard “Tommy” Guy (Microsoft)\n\nEdmund Hart (University of British Columbia)\n\nNeil Chue Hong (Software Sustainability Institute)\n\nKaty Huff (University of Wisconsin)\n\nMichael Jackson (Edinburgh Parallel Computing Centre)\n\nW. Trevor King (freelance software developer)\n\nJustin Kitzes (University of California, Berkeley)\n\nStephen McGough (University of Newcastle)\n\nLex Nederbragt (University of Oslo)\n\nTracy Teal (Michigan State University)\n\nBen Waugh (University College London)\n\nLynne J. Williams (Rotman Research Institute)\n\nEthan White (Utah State University)\n\n\nSupplementary materials\n\n\n\n\nReferences\n\nCook JD: Moore’s Law Squared, 2012. Reference Source\n\nHannay JE, Langtangen HP, MacLeod C, et al.: How do scientists develop and use scientific software? In Second International Workshop on Software Engineering for Computational Science and Engineering (SECSE09). 2009; 1–8. Publisher Full Text\n\nPrabhu P, Jablin TB, Raman A, et al.: A survey of the practice of computational science. In Proceedings of the 24th ACM/IEEE Conference on High Performance Computing, Networking, Storage and Analysis, 2011. Publisher Full Text\n\nWilson G, Aruliah DA, Brown CT, et al.: Best practices for scientific computing. PLoS Biol. 2014; 12(1): e1001745. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAranda J: Software Carpentry Assessment Report, 2012. Reference Source\n\nWilson GV: What should computer scientists teach to physical scientists and engineers? IEEE Computational Science and Engineering. Summer and Fall 1996; 3(2): 46–65. Publisher Full Text\n\nWilson G: Where’s the Real Bottleneck in Scientific Computing? Am. Sci. 2006; 94(1): 5. Publisher Full Text\n\nWilson G: Software carpentry: getting scientists to write better code by making them more productive. Comput Sci Eng. 2006; 8(6): 66–69. Publisher Full Text\n\nJordan K: MOOC completion rates: The data, 2013. Reference Source\n\nSoftware Carpentry web site. Reference Source\n\nSoftware Carpentry github learning materials repository. Reference Source\n\nSoftware Carpentry instructor training. Reference Source\n\nWomen in computing: the gender gap. Reference Source\n\nGuzdial M: Why is it so hard to learn to program? In Andy Oram and Greg Wilson, editors, Making Software: What Really Works, and Why We Believe It. O’Reilly Media, 2010; 111–124. Reference Source\n\nGuzdial M: Exploring hypotheses about media computation. In Proc. Ninth Annual International ACM Conference on International Computing Education Research. ICER’13, 2013; 19–26. Publisher Full Text\n\nHazzan O, Lapidot T, Ragonis N: Guide to Teaching Computer Science: An Activity-Based Approach. Springer, 2011. Publisher Full Text\n\nPorter L, Guzdial M, McDowell C, et al.: Success in introductory programming: What works? Communications of the ACM. 2013; 56(8): 34–36. Publisher Full Text\n\nSorva J: Visual Program Simulation in Introductory Programming Education. PhD thesis, Aalto University, 2012. Reference Source\n\nWilson G: Attendance and number of Software Carpentry workshops January 2012 - January 2014. figshare. 2014. Data Source" }	[ { "id": "3814", "date": "24 Feb 2014", "name": "Juha Sorva", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 insightful and well-written commentary on a timely topic. The article documents the past and present practices of Software Carpentry - a project for teaching scientists about computing - and reflects on the project's successes and failures. In doing so, it provides concrete examples of the teaching practices used as well as those discarded. Moreover, the article helps the reader to understand how teaching scientists to about computing is different from teaching computer science majors - a matter that is central to the efforts of Software Carpentry and to the interests of the growing numbers of scientists who need computing skill to work efficiently.The commentary is well grounded in evidence from the research literature as well as the author's lengthy experience with the project. The achievements and challenges of Software Carpentry are discussed realistically and critically.Additional comment: There is only so much you can learn in two days (the length of Software Carpentry's current workshops), and whatever you learn in that time is unlikely by itself to change your research practices dramatically. What would be interesting to know in the future is whether and how the workshop participants go about building their computing skills after attending a workshop.", "responses": [] }, { "id": "3815", "date": "26 Feb 2014", "name": "Philip 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 article is a retrospective on the past 15 years of the author leading the Software Carpentry effort to educate scientists about the practical value of computational tools. It also describes a set of instructional practices that have worked well (and some that have not worked so well) in this setting. It concludes with ongoing and future work to scale up Software Carpentry in light of large variations in instructor and student backgrounds, and continual changes in modern computational tools.This article is a great fit for F1000 due to the topic's relevance to researchers in life sciences (and across a diverse array of science fields), and to the author's cogent firsthand accounts of his experiences and reflections on a subject matter which he is well-suited to discuss.My main high-level comment is that the language is colloquial in many parts of this article, with lots of asides enclosed (in parentheses). That is probably fine for an opinion-based article, and it makes the writing more personal and approachable. But the author should be aware that this is how the article appears to a first-time reader.Here are some more detailed comments, none of which are pressing:\"From red to green\" -- it took me a while to understand the \"red\", \"orange\", \"green\" light analogy the author was making in this section. That seems to be culturally specific. (I don't think I've seen an orange traffic light.) \"Versions 2 and 3: Another red light\" - I didn't understand why these were two separate versions. Maybe it's simpler just to call this Version 2 and update the subsequent version numbers? \"It works too well to be interesting\" -- This blurb felt a bit harsh toward CS professors. It makes it sound like they teach only topics that lead to new publishable research. In my experience, teaching and research are fairly decoupled, so professors have no qualms about teaching materials from, say, 30-year-old compilers or databases textbooks, which are obviously not leading to new research. Perhaps a more likely explanation, which the author points out later in the article, is that there simply isn't room in CS curricula to offer these sorts of Software Carpentry-like materials, and nobody vouches strongly enough for them. Typo in caption: \"Enrolment figures\" -> \"Enrollment figures\" \"What we do\" - \"Day 1 a.m.\", etc. -- that's hard to parse. I thought the author meant \"1am\" like they were offering a class at 1 in the morning. Same with \"1pm\", \"2am\", \"2pm\". \"Day 1 - morning\" would be clearer. \"during the practical\" - I'm not familiar with this phrase. Is that a typo, or a figure of speech? \"It's a refreshing change from teaching undergraduate calculus.\" -- would Software Carpentry instructors ordinarily teach calculus? Seems more like they would be teaching physics or programming or something.", "responses": [] }, { "id": "3816", "date": "24 Mar 2014", "name": "Hilmar Lapp", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 some of the origins, driving motivations and lessons learned over the more than 15 years of iterative improvements and reboots of Software Carpentry, a brand of (meanwhile) travelling workshops teaching fundamental best practices in software engineering to programming scientists.Software Carpentry has received wide acclaim, and helps fill critical gaps in a time when creating and using computational tools is becoming indispensable to increasingly many scientific fields. As such, the topic is of broad interest without question. The text is well-written, and in most places well argued. My only two overarching critiques are (1) that the author in some places seems to conflate cause and effect; and (2) that in some places I feel the reader is left hanging with a too little information. However, none of these rise to the level of calling into question the validity of the overall conclusions, and thus don't exceed what one might call \"minor revisions\".Since this is an open review, I have chosen to record my detailed comments as public text annotations, using the Hypothes.is (http://hypothes.is) platform, with a transcription also provided below. A PDF version of the comments is also available. The Hypothes.is version of these comments can be accessed at this URL:https://hypothes.is/stream#?user=hlapp&uri=http:%2F%2Ff1000research.com%2Farticles%2F3-62%2Fv1Unfortunately, the ordering of the comments on Hypothes.is appears to be in reverse chronological order (most recent first), and the comments should therefore be read last to first to align with reading the text start to end.Any comments or replies to these comments should be made using the F1000Research ‘Add yours’ option but could also be added to Hypothes.is directly if desired. IntroductionParagraph 1: “hardware and algorithms are only two sides of the iron triangle of programming”Is there a reference for this form of the Iron Triangle? Googling the phrase only turns up the well-known project management Iron Triangle, and its adaptation to software projects. The latter has Resources, Scope, and Time at its corners, not hardware, algorithms, and programming. Paragraph 1: “desktop majority”Do you mean the complement to those doing HPC? The phrase strikes me as needlessly cryptic. And are you sure that scientists developing HPC software are exempt from the trend you describe? Paragraph 2: “rarely if ever shown how to design a maintainable program in a systematic way”Are they not in fact taught, even if only indirectly, that programs are typically not revisited again once passed on (to the course instructor, for example), and hence thinking about maintainability is wasted effort? Paragraph 3: “learning, and applying”Assuming that learning binds to at least some of what we taught as well, the comma is extraneous. Or add a comma after “and applying”. Paragraph 4: “many researchers still find it hard to apply what we teach”Researchers at-large, or researchers who participated in a Software Carpentry workshop?From red to greenVersion 1: Red light Paragraph 1: “i.e., to parallelize complex programs”This seems more an example to me than a restatement of run before they could walk. Thus, this should be “e.g.” (or spelled out “for example”). Paragraph 2: “(then director of the Advanced Computing Laboratory at Los Alamos National Laboratory)”Change parentheses to comma. The parenthetical phrase is important to make sense of the sentence. (And if similar contextual information can be given about Brent Gorda, i.e., information that helps to understand why he was invited, I suggest that be added too, as his current affiliation fails to explain that.) Paragraph 2: “In response, John Reynders (then director of the Advanced Computing Laboratory at Los Alamos National Laboratory) invited the author and Brent Gorda (now at Intel) to teach a week-long course on these topics to LANL staff. The course ran for the first time in July 1998, and was repeated nine times over the next four years.”I suggest that the author highlights the major ways in which these courses differ from the SwC courses run today. As written now, deducing that from the two lessons learned is left as an exercise to the reader, and only those already familiar with SwC will know that indeed today's SwC workshops do differ in these ways. Versions 2 and 3: Another red light Paragraph 2: “(even though a significant minority of their students, particularly those coming from non-CS backgrounds, have no more experience of practical software development than the average physicist”Remove parentheses. Paragraph 2: “In the absence of an institutional mechanism to offer credit courses at some inter-departmental level, this course, like many other interdisciplinary courses, fell between two stools.”Perhaps this would be beyond the scope of the paper as a commentary, but it would be interesting to see whether this is then different at decidedly interdisciplinary programs, for example programs interfacing computational biology / computer science / math. Paragraph 3: “It works too well to be interesting”Based on context, “it” would be the SwC workshop or material. I suggest to reword so it is clear that it actually refers to the practices and tools being taught by SwC. Paragraph 3: “As long as universities reward research first, and supply teaching last, it is simply not in most computer scientists own best interests to offer this kind of course.”If this is the main driver behind this kind of course not finding interest at university CS programs, is the situation then different at teaching-focused schools, such as small liberal arts colleges? There are small liberal arts colleges with strong CS programs; have they indeed been more welcoming to adopting SwC into their curricula? Paragraph 4: “This is partly because educators’ preferred file formats (Word, PowerPoint, and PDF) can’t be handled gracefully by existing version control systems, but more importantly, there simply isn’t a “culture of contribution” in education for projects like Software Carpentry to build on”I'm not convinced that one isn't mostly or entirely a consequence of the other. Open source and collaborative development also was far less widespread in scientific software development before many of the barriers to that were significantly reduced by distributed version control such as Git, and usability and social coding focused resources such as Github. If the tools and file formats that are most widely used are simply refractory to collaboration, it's not a surprise if then a culture of collaboration is rare. Paragraph 7: “The sweet spot for this kind of training is therefore the first two or three years of graduate school. At that point, students have time (at least, more time than they’ll have once they’re faculty) and real problems of their own that they want to solve.”Perhaps it's primarily the “real problems of their own” that provide the motivation for having the time (to learn about addressing them). I.e., percentage-wise, how many students does SwC get today who take the course primarily because they have time, and who do not yet have real problems of their own for which they hope to learn solutions?More importantly perhaps, does this not also point out a path for justifying the inclusion of SwC-inspired teaching units into undergraduate CS curricula? While for some (or most?) academic research career paths the relevance of version control mastery is perhaps less obvious, it's a qualification nearly all of industry ask of CS graduates applying for a software engineer position.Version 4: orange light Paragraph 1: “The author rebooted Software Carpentry in May 2010 with support from Indiana University, Michigan State University, Microsoft, MITACS, Queen Mary University of London, Scimatic, SciNet, SHARCNet, and the UK Met Office.”The backstory to what motivated (or necessitated?) the large consortium of funders is missing here. However, given the last paragraph in this section, it seems there would be interesting aspects of it that would help make setting up the argument. Does the large consortium reflect primarily wide buy-in to SwC's utility, or primarily the difficulty of obtaining enough funding from any one institution or partner? The last paragraph suggests it's the latter, but it's not clear. Paragraph 1: “MOOC”Spell out at first use. Paragraph 2: “Open access publishing, crowd sourcing, and dozens of other innovations had convinced scientists that knowing how to program was now as important to doing science as knowing how to do statistics.”Is there evidence or references for the factors the author enumerates constituting the major driving causes? More specifically, the list is conspicuously missing the explosion of data that had swept, and has continued to sweep into almost every scientific discipline. Data richness is enormously powerful for science, yet wrestling insight from it at this scale invariably and pervasively requires computational processing. Maybe this is part of the “dozens of other innovations”, but I would still argue that the data deluge has constituted a primary rather than a marginal driver of this landscape change. Paragraph 4: “Most importantly, the MOOC format didn’t work”I think it's worth to qualify this statement in respect to the goals. As the paragraph goes on, it could be said that In some definition the MOOC format has worked (for example, compared to retention and completion rates of other MOOCs); the failure that the author reports presumably means chiefly that the goals laid out for a SwC course weren't met by the MOOC format. Paragraph 5: “The biggest take-away from this round was the need come up with a scalable, sustainable model. One instructor simply can’t reach enough people, and cobbling together funding from half a dozen different sources every twelve to eighteen months is a high-risk approach.”For readers who aren't already fully on board with this, It would help to better set up the argument. Why is scaling up the model desirable or necessary? What is enough people? Couldn't funding also come from a single or few sources? Many courses are sustained by student tuition; how would this likely not work for SwC?Version 5: green light Paragraph 1“and backing from the Mozilla Foundation”The difference in wording suggests that the Mozilla Foundation's backing didn't come in the form of a grant. Can it be spelled out (at least broadly) what that support consisted of? Paragraph 1: “This time, the model was two-day intensive workshops”I'm curious as to why 2 days. The lessons learned stated earlier seem to say that attention drops after 3 days, not 2 days. Why was the decision made to shorten to 2 days, not 3 days? Paragraph 1: “The Hacker Within”Is there no link or other reference available? Paragraph 3: “Switching to a “host site covers costs” model was equally important: funding is still needed for the coordinator positions (the author and two part-time administrative assistants at Mozilla, and part of one staff member’s time at the Software Sustainability Institute in the UK), but our other costs now take care of themselves.”I'd find it really useful to spell this out a little more. What are “our other costs”? Instructor travel and expenses, room rental? What tasks do the coordinators perform, how does this scale? Or in other words, presumably there is a division between costs of operating that benefit from economies of scale, and those that do not. More insight into this division would be quite helpful as a lesson learned. Paragraph 4: “have grown steadily (Figure 1 and Figure 2).”The figures suggest a tapering off in the recent past. Is this more likely a fluke due to limited or censored data, or is there a trend showing? Figure 2 : “Enrolment” Typo (one instead of two 'l') Description of Figshare Data: “Hopefully these two effects more or less cancel out and should not detract from the overall trend displayed.”Hope is nice but not a good basis on which to base scientific conclusions. Do you have evidence that suggests that neither fraction of people is significant with respect to those enrolled and attending both days? Evidence that both fractions of people has stayed relatively constant over time, and not changed more recently? Paragraph 5: “80–90% of attendees typically report”What does typically mean? 80-90% of all SwC enrolled students, or on average 80-90% of those enrolled in a workshop? I.e., how much variance is there between workshops? What we do Paragraph 5: “While some people feel that using R instead of Python is like using feet and pounds instead of the metric system”I have heard concerns and objections some people have with R's syntax and way of doing things. But every language (including Python) has its detractors, and I don't think the particular concerns with R are necessarily widely known let alone understood. So I would suggest to either delete this clause (is it really needed for the argument?), or if chosen to be left in place, to substantiate it, at least by giving a reference to a fuller discussion of R's problems. Paragraph 5: “now that we have enough instructors to be able to specialize”It's probably not just a question of having instructors, but also of having demand for (and thus acceptance of) the SwC curriculum as useful in increasingly many disciplinary areas. Paragraph 6: “with the best”Insert \"even\" before “with”. Paragraph 7: “As this is usually several times more than a small registration fee would bring in, we usually choose the higher no-show rate as the lesser evil.”The biggest problem of a significant rate of no-shows is probably the fact that due to the space limitations other students who would have and benefitted from the course had to be denied because of the no-shows taking the space away. Have other possibilities to deter no-shows been explored (and if so, how effective have they been found)?If the no-show rate is somewhat predictable (and it sounds like it is), then wait-listed students could be told to show up anyway on the day of the course, because there would likely be enough no-shows to make room for them. Has this been tried, and to what extent does it work? Paragraph 9: “What does require permission is use of our name and logo, both of which are trademarked. We are happy to give such permission if we have certified the instructor and have a chance to double-check the content, but we do want a chance to check: we have had instances of people calling something “Software Carpentry” when it had nothing to do with what we usually teach. We’ve worked hard to create material that actually helps scientists, and to build some name recognition around it, and we’d like to make sure our name continues to mean something.”This whole paragraph doesn't mention the words \"brand\", \"brand recognition\", and \"brand reputation\"; yet it is essentially about those concepts, isn't it? Why not say it directly?Small things add upUse what we teach Paragraph 1:“The (considerable) downside is that it can be quite difficult for newcomers to contribute material; we are therefore working to streamline that process.”This needs some qualification to fully make sense as following from the preceding sentence. If the tools and approaches SwC teaches are good ones that \"work\", and SwC uses those tools and approaches itself, how can this be a downside, presuming that those able to contribute material are in fact familiar with those tools and approaches. I can imagine some ways in which this can still be a downside, but for clarity this should be spelled out better.Keep experimenting Paragraph 3: “DiRAC”Spell out. Also, how about a URL? Paragraph 5: “Many of our instructors also teach regular university courses, and several of them are now using part or all of our material as the first few lectures in them.”Isn't this somewhat contradicting some lessons learned stated earlier, which seemed to say that for several reasons the SwC curriculum faces impossibly high barriers for integration into university curricula, at least in the current environment. If contrary to expectation this has now become possible, can something be learned from the cases where it has been successfully integrated?TODO Long-term assessment Paragraph 1: “no one knows how to measure the productivity of programmers, or the productivity of scientists”I think this assertion needs better qualification to be really justified. Obviously, several ways to assess programmer productivity, and also scientist productivity, exist. Hiring and tenure committees regularly assess productivity of scientists. Arguably, the ways this is usually done suffers from various problems such as failing to encompass the full spectrum of products resulting from a scientist's work. Perhaps the author means that it is some of these shortcomings of current productivity assessment methods that effectively prevent measuring the productivity impact of SwC's teachings, but that needs to be spelled out better.“Is it supposed to hurt this much?” Paragraph 2:“naive” Is this meant to be \"native\"?Teaching on the web Paragraph 1: “The fact that this is also true of most high-profile MOOCs is little comfort.”If your goal is a high rate of retention and completion, that is. However, widening reach could also be a worthwhile goal. If a single MOOC reaches 10,000 students instead of 800 students reached by 20 physical SwC workshops, even a completion rate of only 10% will still have taught more students with the single MOOC than with the 20 physical workshops. MOOCs clearly aren't a panacea, and they may indeed be ill-suited to the learning objectives of SwC, but that and why this is so needs a little more depth to be convincing. What vs. howParagraph 2: “don’t as good a job”Insert \"do\" after “don't”. Paragraph 2: “xUnit-style framework”I'm embarrassed to ask what's an xUnit style framework. Spell out what that is, and/or add a reference or URL?Standardization vs. customization Paragraph 1: “However, we do need to be more systematic about varying our content on purpose rather than by accident.”As a reader, I feel left hanging by the section ending with this statement. Are there ideas about how this could be done, and to begin with, what were some of the problems encountered with the less systematic approach being practiced now? (The preceding text seems to only cite advantages.)Watching vs. doing Paragraph 1: “We also don’t give them as diverse a range of exercises as we should, and those that we do give are often at the wrong level.” How do you know that this is the case? From feedback alone, or are there other kinds of observations or evidence? Paragraph 2: “though we hope that will diminish as we organize and recruit along disciplinary lines instead of geographically”Aren't you arguing above that diversity of backgrounds and starting skills is a constant challenge? It didn't seem from earlier arguments in the text that simply recruiting along a uniform discipline will address this problem.Better teaching practices Paragraph 1: “We do our best to cover these ideas in our instructor training program, but are less good about actually applying them in our workshops.”Is there some insight available into why instructors find it difficult to apply what they have been taught? Is it the imparting of these ideas that needs improvement, or are the ideas not as applicable in SwC as they were thought to be, or is there simply heterogeneity in that some ideas are much easier to apply than others? If the latter, which ones fall into which category? Conclusions Paragraph 1: “To paraphrase William Gibson”I notice that it's not clear to what exact piece or event to source this. Perhaps still link to the William Gibson Wikiquote page, which includes the quote and its provenance?", "responses": [] } ]	1	https://f1000research.com/articles/3-62
https://f1000research.com/articles/5-115/v1	28 Jan 16	{ "type": "Review", "title": "The critical importance of the fetal hypothalamus-pituitary-adrenal axis", "authors": [ "Charles E. Wood", "Maureen Keller-Wood", "Maureen Keller-Wood" ], "abstract": "The fetal hypothalamus-pituitary-adrenal (HPA) axis is at the center of mechanisms controlling fetal readiness for birth, survival after birth and, in several species, determination of the timing of birth. Stereotypical increases in fetal HPA axis activity at the end of gestation are critical for preparing the fetus for successful transition to postnatal life. The fundamental importance in fetal development of the endogenous activation of this endocrine axis at the end of gestation has led to the use of glucocorticoids for reducing neonatal morbidity in premature infants. However, the choice of dose and repetition of treatments has been controversial, raising the possibility that excess glucocorticoid might program an increased incidence of adult disease (e.g., coronary artery disease and diabetes). We make the argument that because of the critical importance of the fetal HPA axis and its interaction with the maternal HPA axis, dysregulation of cortisol plasma concentrations or inappropriate manipulation pharmacologically can have negative consequences at the beginning of extrauterine life and for decades thereafter.", "keywords": [ "fetal hypothalamus-pituitary-adrenal axis", "HPA", "cortisol" ], "content": "\n\nCortisol is arguably the most important hormone for organizing maturation in late gestation. The discovery that, in sheep, interruption of the fetal hypothalamus-pituitary-adrenal (HPA) axis prevents spontaneous parturition1,2 initiated a series of important studies on the role that cortisol plays in the coordination of fetal readiness for extrauterine life. The mechanism of parturition and the role that the fetal adrenal plays in primate species are complicated compared with ruminants and other large animals. But, although cortisol does not directly initiate parturition in the human, it does have other critically important actions in the human: actions that were first discovered in the sheep model. Perhaps the most impactful discovery was that fetal sheep that were premature but otherwise treated with glucocorticoid were less likely to die in respiratory distress3. The mechanism of this effect involves a direct action of glucocorticoid on the fetal lung, accelerating terminal development and stimulating the production of pulmonary surfactant4–6. This discovery, rapidly translated to the human7,8, has resulted in the widespread antenatal administration of glucocorticoids to pregnant women threatening premature labor9,10. This clinical practice has been an effective strategy for reduction or elimination of ventilator assist of the premature infant after birth. In addition to its effect on the lung, cortisol plays an important role in accelerating development of the fetal gastrointestinal tract11 and liver12. Recent evidence also supports a role for cortisol in the development of the fetal cardiovascular system13 and heart14. Similar to its effect in other systems, cortisol helps ready the fetal cardiovascular system for the transition to extrauterine life.\n\nA central tenet of endocrinology is that circulating concentrations of hormones are controlled: that there is a set point around which negative feedback mechanisms maintain concentrations within a range15,16. For a hormonal system as important as the fetal HPA axis, understanding those mechanisms that govern the increases in fetal adrenocorticotropic hormone (ACTH) and cortisol becomes critically important for understanding the timing of birth, readiness for birth, and survival of the newborn. This most often involves the elucidation of influences, both positive and negative, that determine the trajectory of fetal ACTH secretion. For example, Thorburn and colleagues discovered a dynamic increase in the sensitivity of the fetal adrenal cortex to ACTH that can account for much of the increase in circulating cortisol concentrations prior to birth17. Several laboratories have investigated changes in circulating molecular forms of immunoreactive ACTH (resulting from partial or complete processing of the parent molecule pro-opiomelanocortin), altering the effective biological activity of this pituitary hormone18–21. New evidence suggests that peripheral interconversion of cortisol and cortisone is also an important variable that can contribute to changes in circulating concentrations of cortisol22. Balancing the stimulation of the axis are mechanisms that prevent over-activity of the axis. The late-gestation fetal sheep has a cortisol negative feedback mechanism that is remarkably sensitive to small changes in plasma cortisol concentration23–27. In the final days and hours of fetal life, this negative feedback sensitivity falls dramatically, allowing greater increases in fetal HPA axis activity28.\n\nAlthough nearly every variable within the fetal HPA axis has been studied to some extent, we are not close to fully understanding the interplay between the normal fetal developmental patterns, the influence of fetal and maternal stress, and the modulatory influence of infection on the timing of parturition and the survival of the newborn. A danger in the translation of basic endocrinology to clinical practice has been an under-appreciation of untoward actions of hormones when they are present in unnaturally high concentrations. A clear example of this is the use, in practice, of antenatal steroid administration to women threatening preterm labor. First approved by a National Institutes of Health (NIH) consensus panel as a single treatment or a small number of repeated treatments7, the procedure was modified by practicing physicians so as to administer glucocorticoid weekly from as early as 22 to 24 weeks’ gestation29. Forgetting the basic rule of endocrinology—that control mechanisms prevent excessively high, as well as excessively low concentrations of hormones—can create unintended problems. Weekly or biweekly antenatal maternal betamethasone treatment resulted in the birth of babies that were more likely to be growth-restricted29–31. Studies in animal models indicate that multiple doses of glucocorticoid can have negative neurodevelopmental outcomes. Using the sheep model, Newnham and colleagues clearly demonstrated that excessive treatment with glucocorticoid dramatically impacts fetal brain growth and development32,33. This laboratory and others have also demonstrated negative effects of excessive glucocorticoid on fetal somatic growth34,35. Recent evidence in the baboon model reveals sex-specific effects of antenatal betamethasone on learning and attention in the offspring36. Recent work has suggested that the effects of excess glucocorticoid can be codified in the epigenome of the infant, theoretically with effects that could last a lifetime37,38. On balance, however, the long-term biological cost of multiple treatments is not clear. Clinical trials have not demonstrated long-term growth or major neurosensory disabilities but have indicated an increased likelihood of attention deficit in children who were exposed to multiple doses of glucocorticoid before birth39. Arterial stiffness, however, was increased in 14- to 26-year-old subjects exposed to two to nine weekly doses of betamethasone40. In recognition of the potential risks of multiple antenatal glucocorticoid treatments, a second NIH consensus statement recommended a single treatment41. As a result of the revised recommendations, the incidence of multiple treatment has been reduced.\n\nThe fetus is an organism that is distinct from, yet dependent on, its mother. The circulation of the fetus does not admix with the circulation of the mother, and the fetus in late gestation has the capacity to synthesize its own hormones. However, by virtue of the fact that the fetal and maternal blood is separated by one or more layers of cells in the placenta (depending on the species and therefore the general structure of the placenta), there is a molecular communication between mother and fetus. This communication is most obvious with regard to blood gases. Fetal growth and development depend on the oxygen and nutrients supplied by the mother via trans-placental passage. However, in addition to hormones synthesized within the placenta (e.g., chorionic gonadotropin and chorionic somatomammotropin), there is endocrine communication. An important example of this communication is the influence of maternal adrenal cortical hormones on the fetus, perhaps a mechanism by which the fetus is informed about maternal stress.\n\nThe placentas of many species, including the human, rodent species, and the sheep, express the enzyme 11β-hydroxysteroid dehydrogenase types 1 and 2 (encoded by the genes HSD11B1 and HSD11B2), which interconvert cortisol and cortisone (or, in rodent species, corticosterone and 11-dehydrocorticosterone)42–45. The predominant reaction in placenta is oxidation (cortisol to cortisone and corticosterone to 11-dehydrocorticosterone, mediated by the type 2 enzyme isoform). Because cortisone and 11-dehydrocorticosterone have low affinity for the glucocorticoid and mineralocorticoid receptors (GR and MR, respectively), some of the active glucocorticoid in the maternal circulation is inactivated upon passage into the fetal circulation. Although this is a partial barrier for the natural glucocorticoids, it is not complete. Some of the cortisol that was converted to cortisone upon transplacental passage is converted back to cortisol by the type 1 11β-HSD isoform in target tissues, such as lung46,47, brain48, and heart49–51. As shown in the chronically catheterized fetal sheep model, elevation of maternal cortisol concentration to levels typical of stress increases fetal plasma cortisol concentrations and inhibits fetal ACTH secretion via a negative feedback mechanism52. Synthetic glucocorticoids, such as dexamethasone or betamethasone, are not substrates for 11β-HSD and therefore are not inactivated when passing through the placenta.\n\nThe passage of natural glucocorticoid across placenta from mother to fetus raises the question of whether maternal stress is detrimental to the fetus. There is recent interest in the potential “programming” effects of maternal stress on the pattern of development in the fetus53. There are known glucocorticoid effects on gene methylation and histone modification that can have long-lasting effects on the physiology of the offspring. Perinatal programming of the HPA axis has been reviewed elsewhere54. If “programming” can result from alterations in the genome, however, can it also result from a more immediate effect of hypercortisolemia that bends the arc of development? In other words, what are the more immediate consequences of maternal stress?\n\nThe literature is rife with reports of poor pregnancy outcome when the pregnancy is complicated by maternal stress: low socioeconomic class, partner abuse, and violence55–57. It is not known whether these negative effects on the pregnancy are caused entirely, or even partly, by the maternal cortisol stress response, but there is evidence that suggests that cortisol is at least partly to blame. For example, women with Cushing disease have a higher incidence of stillbirth that is associated with obstructive hypertrophic cardiomyopathy58. The effect of the maternal hypercortisolemia can be reproduced in the sheep model (using cortisol administration rates that mimic the maternal stress response)59. However, maternal cortisol also has an effect on fetal somatic growth, in part secondary to alterations in uterine blood flow60. Increases in maternal cortisol also alter fetal pulmonary and renal fluid balance mechanisms61. How many of these modifications of the pattern of fetal growth and development result in increased incidence of morbidity and mortality in postnatal life? For example, does chronic maternal stress cause small-for-gestation babies62 that are more prone to infant mortality63 or metabolic disease in adulthood? Does cortisol-induced alteration in fetal cardiac development underlie the increased incidence of coronary artery disease in adults who were small for gestational age at birth64?\n\nAs it is detrimental to chronically elevate maternal plasma cortisol concentration, it is important to remember that there is a physiological set point for cortisol in maternal blood during pregnancy, and that reductions in maternal cortisol below that level could be detrimental. Circulating concentrations of cortisol in the human and in other species, such as the sheep, are naturally increased in the latter half of pregnancy65–67. Stress, as previously discussed, further elevates the concentration of cortisol68,69. But the fact that the normal set point for cortisol is increased in pregnancy raises the question of why this occurs and whether there are species differences with regard to mechanism68.\n\nThe normal pregnancy-associated increase in maternal adrenal secretion of cortisol appears to play an important role on both sides of the placenta. Prior to the final stages of fetal life in utero, the majority of the cortisol circulating in the fetal blood derives from the maternal adrenal glands70. Because cortisol plays an important role in developmental processes important for fetal readiness for birth, it is likely that the increase in maternal cortisol plasma concentrations provides an important source of cortisol to the fetus before the fetal HPA axis becomes fully competent. On the other hand, reduction in maternal cortisol concentration below the normal set point, down to the level normally observed in the nonpregnant state, disturbs blood pressure, fluid balance, and uterine blood flow, and slows fetal growth66. Although the above-mentioned studies were performed in sheep, evidence from human pregnancies is consistent: untreated maternal adrenal insufficiency causes premature labor and neonatal morbidity71,72.\n\nIt is important to recognize both the importance and the complexity of the HPA axis during pregnancy and during fetal development. The mechanisms controlling this endocrine axis during pregnancy (why is maternal cortisol normally increased during late gestation?) and fetal life (what causes the increase in fetal HPA axis activity that is important for neonatal survival?) have never been fully solved. For example, is the ontogenetic rise in fetal HPA axis activity caused secondary to the development of immune cells within the fetal brain73? Are there placental signaling molecules (e.g., Corticotropin Releasing Hormone [CRH] in primate species) that stimulate fetal HPA axis activity74? Are there programmed developmental events within the fetal brain that progress without modification by circulating endocrine signals? We argue that answering these basic questions will be important for reducing neonatal mortality and morbidity, for improvement of maternal health, and the design of better and smarter treatments for women threatening preterm labor, and for the babies that are admitted to neonatal intensive care units.", "appendix": "Competing interests\n\n\n\nThe authors declare that they 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\nLiggins GC, Kennedy PC, Holm LW: Failure of initiation of parturition after electrocoagulation of the pituitary of the fetal lamb. Am J Obstet Gynecol. 1967; 98(8): 1080–6. 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Am J Physiol Regul Integr Comp Physiol. 2014; 307(4): R405–13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJensen E, Wood CE, Keller-Wood M: Chronic alterations in ovine maternal corticosteroid levels influence uterine blood flow and placental and fetal growth. Am J Physiol Regul Integr Comp Physiol. 2005; 288(1): R54–61. PubMed Abstract \| Publisher Full Text\n\nJensen E, Wood CE, Keller-Wood M: Alterations in maternal corticosteroid levels influence fetal urine and lung liquid production. J Soc Gynecol Investig. 2003; 10(8): 480–9. PubMed Abstract \| Publisher Full Text\n\nBrunton PJ: Effects of maternal exposure to social stress during pregnancy: consequences for mother and offspring. Reproduction. 2013; 146(5): R175–89. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nMcCormick MC: The contribution of low birth weight to infant mortality and childhood morbidity. N Engl J Med. 1985; 312(2): 82–90. PubMed Abstract \| Publisher Full Text\n\nBarker DJ, Winter PD, Osmond C, et al.: Weight in infancy and death from ischaemic heart disease. Lancet. 1989; 2(8663): 577–80. PubMed Abstract \| Publisher Full Text\n\nCarr BR, Parker CR Jr, Madden JD, et al.: Maternal plasma adrenocorticotropin and cortisol relationships throughout human pregnancy. Am J Obstet Gynecol. 1981; 139(4): 416–22. PubMed Abstract \| Publisher Full Text\n\nJensen E, Wood C, Keller-Wood M: The normal increase in adrenal secretion during pregnancy contributes to maternal volume expansion and fetal homeostasis. J Soc Gynecol Investig. 2002; 9(6): 362–71. PubMed Abstract \| Publisher Full Text\n\nJensen EC, Bennet L, Wood C, et al.: Loss of the pregnancy-induced rise in cortisol concentrations in the ewe impairs the fetal insulin-like growth factor axis. Reprod Fertil Dev. 2011; 23(5): 665–72. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKeller-Wood M: Reflex regulation of hormonal responses during pregnancy. Clin Exp Pharmacol Physiol. 1995; 22(2): 143–51. PubMed Abstract \| Publisher Full Text\n\nKutzler MA, Coksaygan T, Ferguson AD, et al.: Maternally administered dexamethasone at 0.7 of gestation suppresses maternal and fetal pituitary and adrenal responses to hypoxemia in sheep. Pediatr Res. 2004; 55(5): 755–63. PubMed Abstract \| Publisher Full Text\n\nHennessy DP, Coghlan JP, Hardy KJ, et al.: The origin of cortisol in the blood of fetal sheep. J Endocrinol. 1982; 95(1): 71–9. PubMed Abstract \| Publisher Full Text\n\nBjörnsdottir S, Cnattingius S, Brandt L, et al.: Addison's disease in women is a risk factor for an adverse pregnancy outcome. J Clin Endocrinol Metab. 2010; 95(12): 5249–57. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nFux Otta C, Szafryk de Mereshian P, Iraci GS, et al.: Pregnancies associated with primary adrenal insufficiency. Fertil Steril. 2008; 90(4): 1199.e17–20. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nRabaglino MB, Keller-Wood M, Wood CE: Transcriptomics of the late gestation ovine fetal brain: modeling the co-expression of immune marker genes. BMC Genomics. 2014; 15(1): 1001. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcLean M, Bisits A, Davies J, et al.: A placental clock controlling the length of human pregnancy. Nat Med. 1995; 1(5): 460–3. PubMed Abstract \| Publisher Full Text" }	[ { "id": "12166", "date": "28 Jan 2016", "name": "Peter W. Nathanielsz", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12167", "date": "28 Jan 2016", "name": "Charles Ducsay", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12168", "date": "28 Jan 2016", "name": "Dean A. Myers", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-115
https://f1000research.com/articles/5-114/v1	28 Jan 16	{ "type": "Review", "title": "Podocytes", "authors": [ "Jochen Reiser", "Mehmet M. Altintas", "Mehmet M. Altintas" ], "abstract": "Podocytes are highly specialized cells of the kidney glomerulus that wrap around capillaries and that neighbor cells of the Bowman’s capsule. When it comes to glomerular filtration, podocytes play an active role in preventing plasma proteins from entering the urinary ultrafiltrate by providing a barrier comprising filtration slits between foot processes, which in aggregate represent a dynamic network of cellular extensions. Foot processes interdigitate with foot processes from adjacent podocytes and form a network of narrow and rather uniform gaps. The fenestrated endothelial cells retain blood cells but permit passage of small solutes and an overlying basement membrane less permeable to macromolecules, in particular to albumin. The cytoskeletal dynamics and structural plasticity of podocytes as well as the signaling between each of these distinct layers are essential for an efficient glomerular filtration and thus for proper renal function. The genetic or acquired impairment of podocytes may lead to foot process effacement (podocyte fusion or retraction), a morphological hallmark of proteinuric renal diseases. Here, we briefly discuss aspects of a contemporary view of podocytes in glomerular filtration, the patterns of structural changes in podocytes associated with common glomerular diseases, and the current state of basic and clinical research.", "keywords": [ "Podocytes", "kidney glomerulus", "urinary ultrafiltrate", "glomerular filtration" ], "content": "Podocytes and glomerular filtration\n\nPodocytes (or visceral epithelial cells) are terminally differentiated cells lining the outer surface of the glomerular capillaries. As a major component of the ultrafiltration apparatus, podocytes have a complex cellular architecture consisting of cell body, major processes that extend outward from their cell body, forming interdigitated foot processes (FPs) that enwrap the glomerular capillaries1. Major processes are tethered by microtubules and intermediate filaments while FPs contain actin-based cytoskeleton2–4. Podocyte FPs comprise a functioning slit diaphragm (SD) in between5,6, a meshwork of proteins actively participating in podocyte signaling7–9. In addition, FPs have a thick, negatively charged coat (glycocalyx) facing the urinary space10; this accounts for negative surface charges throughout the glomerular filtration barrier, which generates an electrostatic repel between the neighboring FPs and helps maintain the unique cytoarchitecture of podocytes by enhancing the physical separation11. Podocytes form the glomerular filtration barrier together with the opposing monolayers of fenestrated endothelium in the vascular space12 and glomerular basement membrane (GBM) in between13,14. This three-layer filtration barrier serves as a size-selective and charge-dependent molecular sieve facilitating the filtration of cationic molecules, electrolytes, and small and midsized solutes but restricting the passage of anionic molecules and macromolecules15,16. It is important to bear in mind that those layers should be arranged with decreasing selectivity, with the SD being the least selective filter; otherwise, retained plasma proteins would routinely accumulate behind the filtration slits of podocytes17. This elegant structure has to oppose hydrostatic pressure in the glomerular capillary, which is the natural driving force behind macromolecular filtration.\n\nIf podocytes are injured, mutated, or lost, the elaborate structure of podocytes is physically altered—a process termed ‘foot process effacement’, which is found in many proteinuric kidney diseases. In some cases, once FPs are effaced (flattened down and fused), the glomerular filtration barrier is no longer intact as evidently indicated by the massive leak of proteins out of the vasculature into the urine, known as proteinuria18. Proteinuria (also referred to as ‘albuminuria’ or ‘microalbuminuria’) is a clinically important sign of early renal dysfunction. In the following sections, we outline the response of podocytes to various stimuli or injury (or both) to better understand the mechanisms underlying podocyte FP effacement, proteinuria, and glomerular disease progression.\n\n\nMajor causes of podocyte injury\n\nPodocyte function depends on a highly ordered cellular arrangement of filtration compartments and the correct signaling within this microenvironment. Therefore, podocytes are uniquely sensitive to a variety of agents interfering with their actin cytoskeleton, their apical membrane domain (i.e., the negative surface charge), SD complex that regulates podocyte actin reorganization, and GBM structure to which podocytes adhere19–21. The mechanisms leading to podocytopathies at the molecular level include genetic events (genetic mutations and deletions) associated with common complex diseases22,23.\n\nThe core structural component of podocyte FPs is a highly regulated actin cytoskeletal network, which was represented either by a dense bundle of actin filaments that extends along the length of FPs or by a relatively short and branched cortical network, which is located at the cell periphery and anchors elements of the SD. The initial response of podocytes to injury is the disruption of these structures and actin dysregulation, where actin and actin-binding proteins accumulate.\n\nIn experimental models aiming to study various aspects of cell and molecular biology of podocytes, it has been demonstrated that podocytes are the major targets of various soluble and cellular products, including toxins, reactive oxygen species (ROS), complements, and antibodies, as outlined in Table 1.\n\nGBM, glomerular basement membrane.\n\nA commonly used experimental model to induce glomerular proteinuria is puromycin aminonucleoside (PAN) injection into rats. Upon PAN treatment, podocytes undergo significant alterations ranging from FP effacement and cytoskeletal rearrangement to diminished levels of actin cytoskeleton- and SD-associated proteins24–28. Abnormal distribution of SD proteins29 and increasing levels of tight junction proteins6,30,31 are also reported. Rats develop proteinuria after 4 or 5 days. The early phase of proteinuria is related to the secretion of hyposialylated angiopoietin-like 4 (ANGPTL4) from podocytes, which binds avidly to the GBM and is sensitive to glucocorticoids32. Later stages may be mediated by direct oxidative mechanisms. Many pharmacological agents, including dexamethasone33,34, fluvastatin35, erythropoietin analog darbepoetin36, mizoribine37, sialic acid38, and nuclear factor kappa B (NF-κB) inhibitor dehydroxymethylepoxyquinomicin (DHMEQ)39, have been shown to possess the ability to reverse the reorganized stress fiber and cortical actin fiber phenotype observed after PAN treatment.\n\nSimilar structural and functional abnormalities are observed in a rat model of adriamycin (ADR)40–44. However, podocyte cytoskeleton returns to almost normal appearance by day 20 in PAN-treated rats, whereas pathological and functional changes progress and proteinuria sustains during a similar period of time in ADR-induced nephropathy. Of note, most mouse strains are not susceptible to either of these reagents except BALB/c and BALB/cJ mice, which develop severe proteinuria and progressive renal failure following ADR administration45,46. Recently, a nuclear DNA repair protein Prkdc (protein kinase, DNA-activated, catalytic peptide) was discovered to participate in the maintenance of the mitochondrial genome and prevent ADR-induced nephropathy47. Simvastatin48 and thiazolidinedione49 also confer renoprotective phenotype in response to ADR.\n\nOther toxins, which act on podocytes and have been used experimentally, include diphtheria toxin (DT)50–52 secreted by Corynebacterium diphtheria, which causes acute loss of podocytes in inducible diphtheria toxin receptor (iDTR) mice; uremic toxin indoxyl sulfate53, which decreases the expression of podocyte differentiation and functional marker proteins; and hemolytic uremic Shiga toxin54–56, which mediates the release of inflammatory cytokines and vasoactive mediators while potently inhibiting protein synthesis.\n\nThese models represent irreversible glomerular damage with major (20% and above in vivo) podocyte depletion50, which leads to progressive renal failure. If podocyte loss is less than this threshold, then podocytes have the capacity to recover the normal structure of a healthy glomerulus. Administration of lipopolysaccharides (LPS) is an example of such a reversible model57–59. LPS trigger podocyte FP effacement and transient proteinuria within 24 hours, which return to baseline after 3 days58. Podocytes sense LPS by Toll-like receptor 4 (TLR-4) and this pro-inflammatory response upregulates expression of the co-stimulatory molecule B7-157 and the urokinase-type plasminogen activator receptor (uPAR)60. LPS also induces the cytosolic variant of cathepsin L (CatL) enzyme61, indicating that CatL upregulation in podocytes is associated with the development of proteinuria in mice through a mechanism that involves the cleavage of large GTPase dynamin61, synaptopodin62, and CD2-associated protein (CD2AP)63. In a recent study, we reported that the modification of intracellular pH by glutamine uptake was a protective mechanism of cultured mouse podocytes against cytosolic CatL activity, which was markedly elevated under the disease state64. Treatment of cultured human podocytes with TLR-3 immunostimulant polyinosinic-polycytidylic acid (polyIC) induces CatL mRNA and simultaneously downregulates podocyte marker protein synaptopodin65, suggesting that polyIC may follow an injury pathway similar to that of LPS.\n\nAnother means of podocyte injury are subepithelial immune complexes developing as a result of circulating antibodies, which damage or activate podocytes through complement-dependent processes. A number of signaling pathways have been implicated in complement-mediated podocyte injury66–69, in which sublethal concentrations of complement produce a pronounced but reversible disruption of the actin cytoskeleton and associated focal contacts70. Other intracellular events include endoplasmic reticulum (ER) stress, production of ROS, and proteases. Focusing on immunologically induced glomerular injury, podocytes also respond to immunologic processes particularly targeting GBM. Passive administration of heterologous sera containing cross-reacting antibodies against the GBM results in vacuolization of podocytes, focal detachment of podocytes from GBM, and immediate onset of glomerulosclerosis with crescent formation71–78 consistent with a crosstalk between podocytes and the immune system.\n\nDistortion of glomerular charge selectivity by neutralization of the negative charges on podocytes and SDs with polycation protamine sulfate (PS) causes FPs to broaden in vivo30,79–81 and stress fibers to disintegrate in vitro82 in a calcium-dependent manner83,84. These physiological changes happen within 15 minutes following PS treatment and can be reversed by reperfusion with heparin for another 15 minutes81,85,86. PS is also responsible for the phosphorylation of SD protein nephrin81,86 and focal adhesion complex protein Cas81. On the other hand, protamine had little or no effect on the sieving coefficient (also referred to as fractional clearance) of bovine serum albumin once added to neutralize GBM polyanions, a finding that downplays the contribution of GBM to the charge selectivity exhibited by the glomerular filtration barrier87. A similar structural alteration can be induced by polycation poly-L-lysine85, by removal of the sialic acid11,88, or by mutation in glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase (GNE), the rate-limiting enzyme of sialic acid biosynthesis89. Patients with mutations in GNE, however, develop the rare muscle disease HIBM (hereditary inclusion body myopathy) and never get kidney disease89, highlighting the differences between mice and humans in this pathway in the kidney.\n\nMutations, abnormalities, or genetic overexpression or deletion in genes encoding podocyte proteins, which are the regulators of actin cytoskeleton such as synaptopodin (SYNPO)90–92, α-actinin 4 (ACTN4)93–96, dynamin (DYN)61,97,98, aarF domain-containing kinase 4 (ADCK4)99, anillin (ANLN)100, apolipoprotein L1 (APOL1)101, RhoA-activated Rac1 GTPase-activating protein 24 (ARHGAP24)102, Rho guanine nucleotide dissociation inhibitor-α (ARHGDIA)103–105, claudin-1 (CLDN1)106, chloride intracellular channel 5A (CLIC5A)107, cofilin-1 (CFL1)108,109, ezrin (EZR)110, inverted formin 2 (INF2)111–114, kidney ankyrin repeat-containing protein (KANK1, KANK2, KANK4)115, neuronal Wiskott-Aldrich syndrome protein (N-WASP)116, class II phosphoinositide 3-kinase C2 α (PI3KC2α)117, phospholipase C ε1 (PLCE1)118, Rho family small GTP-binding protein Rac1 (RAC1)119,120, rhophilin 1 (RHPN1)121, schwannomin interacting protein 1 (SCHIP1)122, and WT1-interacting protein (WTIP)123, and the ones that are associated with SD complex, including nephrin (NPHS1)124–126, podocin (NPHS2)127–129, CD2-associated protein (CD2AP)63,92,130,131, Nck adaptor protein 1/2 (NCK1, NCK2)86,132,133, transient receptor potential cation channel 6 (TRPC6)134–141, cysteine-rich motor neuron 1 (CRIM1)142, FAT atypical cadherin 1 (FAT1)143, Fyn proto-oncogene (FYN)92,144,145, IQ domain GTPase-activating protein 1 (IQGAP1)146,147, MAGUK Inverted 2 (MAGI-2)148, myosin 1c (MYO1C)149, myosin 1e (MYO1E)150–153, kin of IRRE like 1 (NEPH1)126,154, and zonula occludens 1 (ZO-1)155, leads to proteinuric diseases owing to the disruption of filtration barrier and rearrangement of actin cytoskeleton.\n\nLikewise, glomerular filtration barrier is impaired if the podocyte apical membrane domain proteins maintaining the negative surface charge are lost or transferred including podocalyxin (PC)156,157, protein-tyrosine phosphatase receptor o/glomerular epithelial protein 1 (PTPRO/GLEPP1)158,159, cdc42 (CDC42)119,160, atypical protein kinase Clambda/iota (aPKCλ/ι)161–163, glucosamine uridine diphospho–N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE)89, and Van Gogh-like (planar cell polarity) protein 2 (VANGL2)164–166. Highlighting the importance of glomerular capillary wall assembly, manipulating or deleting the genes implicated in the adhesion of podocytes to GBM components such as integrin α3 (ITGA3)27,167, integrin β1 (ITGB1)168,169, integrin β4 (ITGB4)170, CD9 (CD9)171, CD151 (CD151)172–175, type IV collagen α3/α4/α5 (COL4A3, COL4A4, COL4A5)176–178, discoidin domain receptor 1 (DDR1)179, glypican 5 (GPC5)180, integrin-linked kinase (ILK)168,181,182, laminin β2 (LAMB2)183–186, N-deacetylase/N-sulfotransferase (NDST1)187, RAP1 GTPase-activating protein (RAP1GAP)188, and talin 1 (TLN1)189 causes disorganization of podocyte cytoskeletal architecture, leading to deformation in glomerular filtration.\n\nThe involvement of lysosome membrane protein 2 (SCARB2/LIMP2)190 in the maintenance of podocyte structure and mitochondrial proteins coenzyme Q2 (COQ2)191, coenzyme Q6 (COQ6)192, Mpv17 (MPV17)193, mitochondrial tRNA leucine 1 (MTTL1)194, and prohibitin ring complex subunit prohibitin-2 (PHB2)195 in the redox state of podocyte is reported in genetic studies. Podocyte-specific deletion of autophagy-related 5 (ATG5)196, mammalian target of rapamycin (MTOR)197, prorenin receptor (PRR)198–200, and class III phosphoinositide 3-kinase/vacuolar protein sorting 34 (PIK3C3/VPS34)201,202 disrupts intracellular vesicle trafficking and impairs autophagic flux. Ablation of dendrin (DDN)203 improves renal survival in progressive glomerulosclerosis, whereas knockdown of survivin (BIRC5)204, a member of the inhibitor of apoptosis protein family, and Yes-associated protein (YAP)205,206, a downstream target of Hippo kinases, induces podocyte apoptosis. Podocyte-specific knockout mice for vascular endothelial growth factor A (VEGF-A) demonstrated a key role for VEGF-A signaling for the establishment and maintenance of a normal glomerular filtration barrier207 as well as mesangial cell survival and differentiation208.\n\nA tremendous number of genetic studies were carried out to reveal the biological role of various pathways in podocytes. Regulatory genes, including β-catenin (CTNNB1)209, Notch intracellular domain 1 (ICN1)210, ILK211–213, negative factor (NEF)214,215, Notch’s intracellular domain (NOTCH-IC)216, septin (SEPT7)217, transforming growth factor-β (TGF-β)218, tuberous sclerosis complex 1 (TSC1)219, vpr-binding protein (VPR)215, and wingless-type MMTV integration site family 1 (WNT1)220, are used as genetic switches to turn on (activate) a specific signaling pathway. There are also studies aiming to shut down (repress) a particular pathway targeting the genes such as Akt2 (AKT2)221, PINCH-1–binding ankyrin repeat domain of ILK (ANK)222, angiotensin II receptor 2 (AT2)223, CTNNB1209,224,225, diaphanous interacting protein (DIP)226, dickkopf WNT signaling pathway inhibitor 1 (DKK1)209,220,225, insulin-like growth factor-I receptor (IGF-IR)195,227–229, insulin receptor (INSR)195,227,229, NF-κB essential modulator (NEMO)230,231, Notch-1 (NOTCH1)232, Notch-3 (NOTCH3)233, 3-phosphoinositide-dependent kinase-1 (PDK1)234, rapamycin-sensitive adaptor protein of mTOR (RAPTOR)219,235 and rapamycin-insensitive subunit of mTOR (RICTOR)221,235, recombining binding protein suppressor of hairless (RBPSUH)216, SH2-domain-containing inositol polyphosphate 5-phosphatase 2 (SHIP2)236, SMAD family member 2/3 (SMAD2, SMAD3)237, and signal transducer and activator of transcription 3 (STAT3)238–240.\n\nCurrently, there is great interest in research into transcriptional regulation of gene expression patterns during development and differentiation of podocytes241. Genetic studies and analysis of mutations in genes encoding transcription factors provide a comprehensive approach in characterizing the functional role of transcription factors. Alterations in c-Maf-inducing protein (CMIP)242,243, forkhead box C2 (FOXC2)244, hypoxia-inducible factor 1 α (HIF1A)245–247, Krüppel-like factor 6 (KLF6)248, LIM homeobox transcription factor 1 β (LMX1B)249–254, v-Maf avian musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB)255,256, nuclear factor of activated T cells (NFAT)257,258, paired box gene 2 (PAX2)259, podocyte-expressed 1/transcription factor 21 (POD1/TCF21)260, peroxisome proliferator-activated receptor-α (PPARA)261, Snail family zinc finger 1 (SNAI1)262,263, Wilm’s tumor 1 (WT-1)264–268, zinc finger E-box-binding homeobox 2 (ZEB2)269, and zinc fingers and homeoboxes 1/2/3 (ZHX1, ZHX2, and ZHX3)270 were studied to enforce a particular cell fate by stimulating or suppressing the related genes.\n\nIn addition to these molecules, factors, and genes implicating various means of podocyte injury, there are proteins or agents such as angiopoietin-like 3 (ANGPTL3)271, B7-1 (CD80)57,272, cytosolic CatL61–64, glucose273–277, glutamine64, insulin227, integrin β3 (ITGB3)60,278, tumor necrosis factor-α (TNF-α)279–281, transient receptor potential cation channel 5/6 (TRPC5 and TRPC6)282, and uPAR60 that are involved in the regulation of podocyte cytoskeleton. In some cases, this cytoskeletal disaggregation and the associated activation of certain pathways (including tumor suppressor protein p53 and caspases) lead to podocyte loss (in vitro) and detachment from GBM (in vivo). Seminal studies have shown that apoptotic stimuli are mediated by albumin283–285, aldosterone286–288, angiopoietin-like3 (ANGPTL3)289, angiotensin II290–294, fatty acids295–297, glucose232,294,298, IGF-binding protein-3 (IGFBP-3)299, oxidized low-density lipoprotein (LDL)300, and TGF-β1237,301–304. Angiotensin II305 and glucose306 might also induce podocyte autophagic processes as evidenced by the presence of the increased number of autophagosomes and autophagic genes such as LC3-2 and beclin-1. Under specific conditions, podocyte injury leads to a phenotypic conversion, where podocytes lose their epithelial features such as nephrin, P-cadherin, and ZO-1 while acquiring mesenchymal markers such as desmin, fibroblast-specific protein-1 (FSP-1), α-smooth muscle actin (α-SMA), vimentin, type I collagen, and fibronectin307. This process is referred to as podocyte’s epithelial-mesenchymal transition (EMT) and is driven in some cases by endothelin-1 (ET-1)263, ILK212, PAN262, and TGF-β1308. When infected by human immunodeficiency virus 1 (HIV-1) NEF protein309,310 or treated by TNF-α311, the podocyte gives a proliferative response marked by the loss of differentiation markers such as synaptopodin, WT-1, and GLEPP-1 and the subsequent expression of the proliferation markers such as podocyte G1 cyclin, cyclin A, cyclin D1, and Ki-67. There is evidence that non-cytokine-soluble factors such as soluble urokinase-type plasminogen activator receptor (suPAR)278 cause podocyte FP effacement and proteinuria via a β3 integrin-dependent mechanism but that circulating sialylated ANGPTL4312 reduces proteinuria via an endothelial β5 integrin-dependent mechanism. By contrast, podocyte-secreted hypo-sialylated ANGPTL4 causes proteinuria via interactions with the GBM32.\n\nAlthough these complex regulatory mechanisms imply the vulnerability of podocytes, a variety of factors support podocyte differentiation and survival, including activated protein C (APC)313,314, bone morphogenetic protein-7 (BMP7)277,299,315, insulin-like growth factor-II (IGF-II)228, VEGF-A316–318, and vascular endothelial growth factor C (VEGF-C)318,319.\n\n\nPodocytes in glomerular disease pathology\n\nAlthough the podocyte injury is not the only cause of major glomerular diseases, a stable podocyte architecture with interdigitating FPs connected by highly specialized filtration slits is essential for the maintenance and proper function of the glomerular filtration barrier. Both experimental and clinical studies have indicated a pivotal role of podocyte injury in the development and progression of glomerular diseases.\n\nA number of different conditions and health risk factors can result in glomerular disease. Nephrotic syndrome or glomerulonephritis (i.e., malfunction of glomerular filter) may be a direct result of an infection or accumulation of toxic agents in kidneys, or (podocyte- and GBM-associated) genetic defects, or may be due to a secondary insult such as a pre-existing disease occurring in the body320–322. This represents the conventional approach to classification of glomerular diseases, which generally meets the needs of nephrologists.\n\nThe most common cause of primary glomerular disease in adults is focal segmental glomerulosclerosis (FSGS), which is defined by the scarring (sclerosis) of some but not all of the glomeruli (focal) that involves only a section of the affected glomeruli (segmental) by light microscopy of a renal biopsy specimen. In most cases, distinguishing primary (idiopathic) FSGS from the genetic form of FSGS associated with mutations in essential podocyte proteins323,324 or secondary FSGS (linked to a variety of conditions, including viral infections, drug toxicity, or previous glomerular injury)325 is challenging; however, it has been proven that this heterogeneous lesion results from podocyte injury20,50,326,327. Once the integrity of podocyte FPs is lost, podocytes start to detach from underlying GBM at certain sites revealing bare areas of glomerular capillary surface. Later, these bare areas of GBM contact the Bowman’s capsule and form synechia, which represents the earliest committed FSGS lesion. This sequence of pathological events eventually leads to the development of more lesions and progression to glomerulosclerosis328. Recurrence of FSGS in renal transplant recipients has given rise to the existence of permeability or circulating factor(s) acting on podocytes as the cause of primary FSGS329. To date, a few plasma factors have been proposed330 but most of these have been found to be non-specific to FSGS serum/plasma331. Recently, suPAR was found to be associated with FSGS; for example, two thirds of patients with primary FSGS exhibited high levels of suPAR and those with the highest levels had a greater chance of recurrence after transplantation278. In support of this, our group found that higher suPAR levels at baseline are independently associated with faster decline in eGFR and suPAR in plasma can predict risk of developing chronic kidney disease (CKD) in healthy people up to five years before its onset332.\n\nContrary to FSGS, in which podocytes are lost in the areas of sclerosis, minimal change disease (MCD) is a reversible disorder with normal histology and does not cause podocyte depletion. Diffuse effacement of podocyte FPs (accompanied by condensation of the actin-based cytoskeleton but not associated with reduction of any key podocyte-specific protein except podocyte alpha-dystroglycan333) and loss of GBM charge are among the classic features of MCD. All of these changes, and the development of selective proteinuria, are attributed to the secretion from podocytes of a form of ANGPTL4 that lacks sialic acid residues32,334.\n\nAn FSGS-related but morphologically distinct phenotype was observed when podocytes are infected with HIV335,336 or induced by infections, drugs, autoimmune diseases, or organ transplants337–339. This phenotype is described as collapsing glomerulopathy (CG) and is characterized by extensive loss of mature podocyte markers, severe FP effacement, and focal detachment together with the collapse of the capillary loops. Importantly, podocytes re-enter the cell cycle, become capable of proliferating, and lead to the formation of crescents filling the Bowman’s space, making CG structurally distinct from other forms of FSGS214,309,340. If left untreated, HIV-1-associated nephropathy progresses to end-stage renal disease within weeks to months, whereas the combined antiretroviral therapy, which blocks HIV-1 replication, limits podocyte hyperplasia and hypertrophy and brings podocytes back to differentiation state341. Studies using animal models have demonstrated that CG can be ameliorated by using cell-cycle inhibitors342 or by activating transcription factors involved in podocyte differentiation343; however, full recovery from CG is scarce344.\n\nThe immunoglobulin A (IgA) nephropathy (IgAN), which is the most prevalent primary chronic glomerular disease worldwide345, exhibits significant heterogeneity in terms of histopathologic features and clinical outcomes346. Emerging data suggest that mesangial deposition of IgA1 immune complexes leads to podocyte necrosis and detachment from the GBM347,348 with the subsequent reduction in nephrin mRNA349.\n\nObesity-related hypertension and diabetes have become epidemic health problems worldwide and major risk factors for the development of CKD350. High glucose altered podocyte actin assembly in vitro351, high blood glucose (hyperglycemia) induced podocyte apoptosis via the ROS-dependent pathway in obese rodents298, and podocyte density and number decreased in patients with obesity-related glomerulopathy352.\n\n\nTargeting podocytes as renal-specific therapy\n\nHuman kidney has been considered a terminally differentiated organ with minimal cellular turnover and limited capacity for repair, suggesting that kidney injuries carrying severe consequences have limited treatment options. The goal of clinical nephrologists and renal researchers should be to identify the renal protection mechanism and to develop strategies for the treatment of kidney or various renal compartments of which kidney is composed. Podocytes are probably the most likely candidate cell population to be analyzed on a molecular level since these intricate cells are the most vulnerable component of the glomerular filtration network even during early stages of injury and serve as hallmarks of a state of glomerular disease353. Owing to their post-mitotic nature, podocytes have a limited capacity for cell division and do not regenerate in response to injury and loss354. This leads to rapid progression of glomerular diseases unless treated. Regardless of the diverse origins of glomerular diseases, podocytes are critical determinants of outcome for all glomerular diseases, which makes podocytes a unique model for monitoring and investigating disease progression355.\n\nTherefore, there has been a pronounced shift toward podocyte proteins as therapeutic targets in the last decade356. Sialic acid and its precursors show efficacy in MCD32 and diabetic nephropathy357. Mutant forms of human ANGPTL4 reduce proteinuria without causing hypertriglyceridemia in FSGS and diabetic nephropathy312,357. Calcineurin inhibitor cyclosporine A (CsA) stabilizes of the actin cytoskeleton and stress fibers in podocytes by blocking the calcineurin-mediated phosphorylation and CatL-facilitated degradation of synaptopodin62. As mentioned earlier, suPAR, which activates integrin αvβ3 independent of uPAR, has been suggested as an FSGS factor278. A specific inhibitor of integrin αvβ3, cyclo-RGDfV, ameliorates proteinuria in mouse models of nephrotic syndrome by directly targeting the upregulated integrin αvβ3 on podocytes60. CD20 antibody rituximab binds to sphingomyelin phosphodiesterase acid-like 3b (SMPDL-3b) and stabilizes the structure and function of podocytes treated with the sera of patients with recurrent FSGS358. Abatacept blocks the interaction of B7-1 (CD80) with cytoskeletal protein talin and thereby stabilizes β1-integrin activity and prevents podocyte motility359. However, these results have been subjected to criticism since uncertainties still remain, preventing us from being too optimistic about the general efficacy of abatacept360–362. The GTPase dynamin, which promotes endocytosis and regulates actin cytoskeleton61,97, is induced by small-molecule Bis-T-23 as a potential therapeutic approach363. Bis-T-23 effectively promotes dynamin assembly into higher-order structures and increases actin polymerization in injured podocytes361.\n\nMolecular analysis of podocytes will lead to a better understanding of disease mechanisms and therefore may enable the identification of targets for early-onset diagnostics and disease treatment. In this context, cell-based high-throughput drug screening assays quantifying the phenotypic changes in podocytes (specifically changes in morphology, F-actin cytoskeleton, focal adhesions, cell volume, and so on) offer great value for the discovery of chemotherapeutic agents. Recently, a podocyte cell-based phenotypic assay was developed and applied to identify novel podocyte-protective small molecules and establish specific drug delivery strategies364.\n\nThe possibilities of targeting podocytes and thereby affecting kidney disease and progression early in the course set high expectations and hopefully will provide a significant benefit to human health in the future.", "appendix": "Competing interests\n\n\n\nJochen Reiser has pending and issued patents on novel strategies for kidney therapeutics and stands to gain royalties from their commercialization. He is co-founder of TRISAQ (Miami, FL, USA), a biotechnology company in which he has financial interest, including stock. MA declares that he has 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\nPavenstädt H, Kriz W, Kretzler M: Cell biology of the glomerular podocyte. Physiol Rev. 2003; 83(1): 253–307. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim JH, Mukherjee A, Madhavan SM, et al.: WT1-interacting protein (Wtip) regulates podocyte phenotype by cell-cell and cell-matrix contact reorganization. Am J Physiol Renal Physiol. 2012; 302(1): F103–15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKestilä M, Lenkkeri U, Männikkö M, et al.: Positionally cloned gene for a novel glomerular protein--nephrin--is mutated in congenital nephrotic syndrome. Mol Cell. 1998; 1(4): 575–82. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nPutaala H, Soininen R, Kilpeläinen P, et al.: The murine nephrin gene is specifically expressed in kidney, brain and pancreas: inactivation of the gene leads to massive proteinuria and neonatal death. Hum Mol Genet. 2001; 10(1): 1–8. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nGarg P, Verma R, Nihalani D, et al.: Neph1 cooperates with nephrin to transduce a signal that induces actin polymerization. Mol Cell Biol. 2007; 27(24): 8698–712. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBoute N, Gribouval O, Roselli S, et al.: NPHS2, encoding the glomerular protein podocin, is mutated in autosomal recessive steroid-resistant nephrotic syndrome. Nat Genet. 2000; 24(4): 349–54. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nHuber TB, Simons M, Hartleben B, et al.: Molecular basis of the functional podocin-nephrin complex: mutations in the NPHS2 gene disrupt nephrin targeting to lipid raft microdomains. Hum Mol Genet. 2003; 12(24): 3397–405. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nRoselli S, Heidet L, Sich M, et al.: Early glomerular filtration defect and severe renal disease in podocin-deficient mice. Mol Cell Biol. 2004; 24(2): 550–60. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nShih NY, Li J, Karpitskii V, et al.: Congenital nephrotic syndrome in mice lacking CD2-associated protein. Science. 1999; 286(5438): 312–5. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nKim JM, Wu H, Green G, et al.: CD2-associated protein haploinsufficiency is linked to glomerular disease susceptibility. Science. 2003; 300(5623): 1298–300. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nJones N, Blasutig IM, Eremina V, et al.: Nck adaptor proteins link nephrin to the actin cytoskeleton of kidney podocytes. Nature. 2006; 440(7085): 818–23. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nJones N, New LA, Fortino MA, et al.: Nck proteins maintain the adult glomerular filtration barrier. J Am Soc Nephrol. 2009; 20(7): 1533–43. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHofstra JM, Lainez S, van Kuijk WH, et al.: New TRPC6 gain-of-function mutation in a non-consanguineous Dutch family with late-onset focal segmental glomerulosclerosis. Nephrol Dial Transplant. 2013; 28(7): 1830–8. PubMed Abstract \| Publisher Full Text\n\nReiser J, Polu KR, Möller CC, et al.: TRPC6 is a glomerular slit diaphragm-associated channel required for normal renal function. Nat Genet. 2005; 37(7): 739–44. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nWinn MP, Conlon PJ, Lynn KL, et al.: A mutation in the TRPC6 cation channel causes familial focal segmental glomerulosclerosis. Science. 2005; 308(5729): 1801–4. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nMöller CC, Wei C, Altintas MM, et al.: Induction of TRPC6 channel in acquired forms of proteinuric kidney disease. J Am Soc Nephrol. 2007; 18(1): 29–36. PubMed Abstract \| Publisher Full Text\n\nHeeringa SF, Möller CC, Du J, et al.: A novel TRPC6 mutation that causes childhood FSGS. PLoS One. 2009; 4(11): e7771. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRocque BL, Babayeva S, Li J, et al.: Deficiency of the planar cell polarity protein Vangl2 in podocytes affects glomerular morphogenesis and increases susceptibility to injury. J Am Soc Nephrol. 2015; 26(3): 576–86. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKreidberg JA, Donovan MJ, Goldstein SL, et al.: Alpha 3 beta 1 integrin has a crucial role in kidney and lung organogenesis. Development. 1996; 122(11): 3537–47. PubMed Abstract\n\nKanasaki K, Kanda Y, Palmsten K, et al.: Integrin beta1-mediated matrix assembly and signaling are critical for the normal development and function of the kidney glomerulus. Dev Biol. 2008; 313(2): 584–93. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nPozzi A, Jarad G, Moeckel GW, et al.: Beta1 integrin expression by podocytes is required to maintain glomerular structural integrity. Dev Biol. 2008; 316(2): 288–301. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nKambham N, Tanji N, Seigle RL, et al.: Congenital focal segmental glomerulosclerosis associated with beta4 integrin mutation and epidermolysis bullosa. Am J Kidney Dis. 2000; 36(1): 190–6. PubMed Abstract \| Publisher Full Text\n\nBlumenthal A, Giebel J, Warsow G, et al.: Mechanical stress enhances CD9 expression in cultured podocytes. Am J Physiol Renal Physiol. 2015; 308(6): F602–13. PubMed Abstract \| Publisher Full Text\n\nKaramatic Crew V, Burton N, Kagan A, et al.: CD151, the first member of the tetraspanin (TM4) superfamily detected on erythrocytes, is essential for the correct assembly of human basement membranes in kidney and skin. Blood. 2004; 104(8): 2217–23. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nSachs N, Kreft M, van den Bergh Weerman MA, et al.: Kidney failure in mice lacking the tetraspanin CD151. J Cell Biol. 2006; 175(1): 33–9. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nBaleato RM, Guthrie PL, Gubler MC, et al.: Deletion of CD151 results in a strain-dependent glomerular disease due to severe alterations of the glomerular basement membrane. Am J Pathol. 2008; 173(4): 927–37. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nBlumenthal A, Giebel J, Ummanni R, et al.: Morphology and migration of podocytes are affected by CD151 levels. Am J Physiol Renal Physiol. 2012; 302(10): F1265–77. PubMed Abstract \| Publisher Full Text\n\nHudson BG, Tryggvason K, Sundaramoorthy M, et al.: Alport's syndrome, Goodpasture's syndrome, and type IV collagen. N Engl J Med. 2003; 348(25): 2543–56. PubMed Abstract \| Publisher Full Text\n\nHudson BG: The molecular basis of Goodpasture and Alport syndromes: beacons for the discovery of the collagen IV family. J Am Soc Nephrol. 2004; 15(10): 2514–27. 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J Am Soc Nephrol. 2011; 22(5): 849–58. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen YM, Zhou Y, Go G, et al.: Laminin β2 gene missense mutation produces endoplasmic reticulum stress in podocytes. J Am Soc Nephrol. 2013; 24(8): 1223–33. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nSugar T, Wassenhove-McCarthy DJ, Esko JD, et al.: Podocyte-specific deletion of NDST1, a key enzyme in the sulfation of heparan sulfate glycosaminoglycans, leads to abnormalities in podocyte organization in vivo. Kidney Int. 2014; 85(2): 307–18. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nPotla U, Ni J, Vadaparampil J, et al.: Podocyte-specific RAP1GAP expression contributes to focal segmental glomerulosclerosis-associated glomerular injury. J Clin Invest. 2014; 124(4): 1757–69. 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J Clin Invest. 2010; 120(4): 1084–96. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nCinà DP, Onay T, Paltoo A, et al.: Inhibition of MTOR disrupts autophagic flux in podocytes. J Am Soc Nephrol. 2012; 23(3): 412–20. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nRiediger F, Quack I, Qadri F, et al.: Prorenin receptor is essential for podocyte autophagy and survival. J Am Soc Nephrol. 2011; 22(12): 2193–202. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nOshima Y, Kinouchi K, Ichihara A, et al.: Prorenin receptor is essential for normal podocyte structure and function. J Am Soc Nephrol. 2011; 22(12): 2203–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi C, Siragy HM: (Pro)renin receptor regulates autophagy and apoptosis in podocytes exposed to high glucose. Am J Physiol Endocrinol Metab. 2015; 309(3): E302–10. 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J Am Soc Nephrol. 2006; 17(3): 724–35. PubMed Abstract \| Publisher Full Text\n\nKato H, Gruenwald A, Suh JH, et al.: Wnt/β-catenin pathway in podocytes integrates cell adhesion, differentiation, and survival. J Biol Chem. 2011; 286(29): 26003–15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNiranjan T, Bielesz B, Gruenwald A, et al.: The Notch pathway in podocytes plays a role in the development of glomerular disease. Nat Med. 2008; 14(3): 290–8. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nKretzler M, Teixeira VP, Unschuld PG, et al.: Integrin-linked kinase as a candidate downstream effector in proteinuria. FASEB J. 2001; 15(10): 1843–5. PubMed Abstract \| Publisher Full Text\n\nTeixeira Vde P, Blattner SM, Li M, et al.: Functional consequences of integrin-linked kinase activation in podocyte damage. Kidney Int. 2005; 67(2): 514–23. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWasik AA, Polianskyte-Prause Z, Dong MQ, et al.: Septin 7 forms a complex with CD2AP and nephrin and regulates glucose transporter trafficking. Mol Biol Cell. 2012; 23(17): 3370–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchiffer M, Bitzer M, Roberts IS, et al.: Apoptosis in podocytes induced by TGF-beta and Smad7. J Clin Invest. 2001; 108(8): 807–16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nInoki K, Mori H, Wang J, et al.: mTORC1 activation in podocytes is a critical step in the development of diabetic nephropathy in mice. J Clin Invest. 2011; 121(6): 2181–96. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nDai C, Stolz DB, Kiss LP, et al.: Wnt/beta-catenin signaling promotes podocyte dysfunction and albuminuria. J Am Soc Nephrol. 2009; 20(9): 1997–2008. 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PubMed Abstract \| Publisher Full Text\n\nJiang L, Xu L, Song Y, et al.: Calmodulin-dependent protein kinase II/cAMP response element-binding protein/Wnt/β-catenin signaling cascade regulates angiotensin II-induced podocyte injury and albuminuria. J Biol Chem. 2013; 288(32): 23368–79. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLu T, He JC, Wang ZH, et al.: HIV-1 Nef disrupts the podocyte actin cytoskeleton by interacting with diaphanous interacting protein. J Biol Chem. 2008; 283(13): 8173–82. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWelsh GI, Hale LJ, Eremina V, et al.: Insulin signaling to the glomerular podocyte is critical for normal kidney function. Cell Metab. 2010; 12(4): 329–40. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nHale LJ, Welsh GI, Perks CM, et al.: Insulin-like growth factor-II is produced by, signals to and is an important survival factor for the mature podocyte in man and mouse. J Pathol. 2013; 230(1): 95–106. PubMed Abstract \| Publisher Full Text\n\nHale LJ, Hurcombe J, Lay A, et al.: Insulin directly stimulates VEGF-A production in the glomerular podocyte. Am J Physiol Renal Physiol. 2013; 305(2): F182–8. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nBrähler S, Ising C, Hagmann H, et al.: Intrinsic proinflammatory signaling in podocytes contributes to podocyte damage and prolonged proteinuria. Am J Physiol Renal Physiol. 2012; 303(10): F1473–85. PubMed Abstract \| Publisher Full Text\n\nBrähler S, Ising C, Barrera Aranda B, et al.: The NF-κB essential modulator (NEMO) controls podocyte cytoskeletal dynamics independently of NF-κB. Am J Physiol Renal Physiol. 2015; 309(7): F617–26. PubMed Abstract \| Publisher Full Text\n\nLin CL, Wang FS, Hsu YC, et al.: Modulation of notch-1 signaling alleviates vascular endothelial growth factor-mediated diabetic nephropathy. Diabetes. 2010; 59(8): 1915–25. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEl Machhour F, Keuylian Z, Kavvadas P, et al.: Activation of Notch3 in Glomeruli Promotes the Development of Rapidly Progressive Renal Disease. J Am Soc Nephrol. 2015; 26(7): 1561–75. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSaurus P, Kuusela S, Lehtonen E, et al.: Podocyte apoptosis is prevented by blocking the Toll-like receptor pathway. Cell Death Dis. 2015; 6: e1752. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGödel M, Hartleben B, Herbach N, et al.: Role of mTOR in podocyte function and diabetic nephropathy in humans and mice. J Clin Invest. 2011; 121(6): 2197–209. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHyvönen ME, Saurus P, Wasik A, et al.: Lipid phosphatase SHIP2 downregulates insulin signalling in podocytes. Mol Cell Endocrinol. 2010; 328(1–2): 70–9. PubMed Abstract \| Publisher Full Text\n\nDas R, Xu S, Quan X, et al.: Upregulation of mitochondrial Nox4 mediates TGF-β-induced apoptosis in cultured mouse podocytes. Am J Physiol Renal Physiol. 2014; 306(2): F155–67. PubMed Abstract \| Publisher Full Text\n\nFeng X, Lu TC, Chuang PY, et al.: Reduction of Stat3 activity attenuates HIV-induced kidney injury. J Am Soc Nephrol. 2009; 20(10): 2138–46. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDai Y, Gu L, Yuan W, et al.: Podocyte-specific deletion of signal transducer and activator of transcription 3 attenuates nephrotoxic serum-induced glomerulonephritis. Kidney Int. 2013; 84(5): 950–61. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbkhezr M, Dryer SE: STAT3 regulates steady-state expression of synaptopodin in cultured mouse podocytes. Mol Pharmacol. 2015; 87(2): 231–9. PubMed Abstract \| Publisher Full Text\n\nChugh SS: Transcriptional regulation of podocyte disease. Transl Res. 2007; 149(5): 237–42. 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PubMed Abstract \| Publisher Full Text\n\nSteenhard BM, Isom K, Stroganova L, et al.: Deletion of von Hippel-Lindau in glomerular podocytes results in glomerular basement membrane thickening, ectopic subepithelial deposition of collagen {alpha}1{alpha}2{alpha}1(IV), expression of neuroglobin, and proteinuria. Am J Pathol. 2010; 177(1): 84–96. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDing M, Cui S, Li C, et al.: Loss of the tumor suppressor Vhlh leads to upregulation of Cxcr4 and rapidly progressive glomerulonephritis in mice. Nat Med. 2006; 12(9): 1081–7. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nMallipattu SK, Horne SJ, D'Agati V, et al.: Krüppel-like factor 6 regulates mitochondrial function in the kidney. J Clin Invest. 2015; 125(3): 1347–61. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nChen H, Lun Y, Ovchinnikov D, et al.: Limb and kidney defects in Lmx1b mutant mice suggest an involvement of LMX1B in human nail patella syndrome. Nat Genet. 1998; 19(1): 51–5. PubMed Abstract \| Publisher Full Text\n\nMorello R, Zhou G, Dreyer SD, et al.: Regulation of glomerular basement membrane collagen expression by LMX1B contributes to renal disease in nail patella syndrome. Nat Genet. 2001; 27(2): 205–8. PubMed Abstract \| Publisher Full Text\n\nMiner JH, Morello R, Andrews KL, et al.: Transcriptional induction of slit diaphragm genes by Lmx1b is required in podocyte differentiation. J Clin Invest. 2002; 109(8): 1065–72. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRohr C, Prestel J, Heidet L, et al.: The LIM-homeodomain transcription factor Lmx1b plays a crucial role in podocytes. J Clin Invest. 2002; 109(8): 1073–82. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSuleiman H, Heudobler D, Raschta AS, et al.: The podocyte-specific inactivation of Lmx1b, Ldb1 and E2a yields new insight into a transcriptional network in podocytes. Dev Biol. 2007; 304(2): 701–12. PubMed Abstract \| Publisher Full Text\n\nBurghardt T, Kastner J, Suleiman H, et al.: LMX1B is essential for the maintenance of differentiated podocytes in adult kidneys. J Am Soc Nephrol. 2013; 24(11): 1830–48. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSadl V, Jin F, Yu J, et al.: The mouse Kreisler (Krml1/MafB) segmentation gene is required for differentiation of glomerular visceral epithelial cells. Dev Biol. 2002; 249(1): 16–29. PubMed Abstract \| Publisher Full Text\n\nMoriguchi T, Hamada M, Morito N, et al.: MafB is essential for renal development and F4/80 expression in macrophages. Mol Cell Biol. 2006; 26(15): 5715–27. 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PubMed Abstract \| Publisher Full Text\n\nGuo JK, Menke AL, Gubler MC, et al.: WT1 is a key regulator of podocyte function: reduced expression levels cause crescentic glomerulonephritis and mesangial sclerosis. Hum Mol Genet. 2002; 11(6): 651–9. PubMed Abstract \| Publisher Full Text\n\nPatek CE, Fleming S, Miles CG, et al.: Murine Denys-Drash syndrome: evidence of podocyte de-differentiation and systemic mediation of glomerulosclerosis. Hum Mol Genet. 2003; 12(18): 2379–94. PubMed Abstract \| Publisher Full Text\n\nChau YY, Brownstein D, Mjoseng H, et al.: Acute multiple organ failure in adult mice deleted for the developmental regulator Wt1. PLoS Genet. 2011; 7(12): e1002404. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHall G, Gbadegesin RA, Lavin P, et al.: A novel missense mutation of Wilms' Tumor 1 causes autosomal dominant FSGS. J Am Soc Nephrol. 2015; 26(4): 831–43. PubMed Abstract \| Publisher Full Text\n\nKumar PA, Kotlyarevska K, Dejkhmaron P, et al.: Growth hormone (GH)-dependent expression of a natural antisense transcript induces zinc finger E-box-binding homeobox 2 (ZEB2) in the glomerular podocyte: a novel action of gh with implications for the pathogenesis of diabetic nephropathy. J Biol Chem. 2010; 285(41): 31148–56. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu G, Clement LC, Kanwar YS, et al.: ZHX proteins regulate podocyte gene expression during the development of nephrotic syndrome. J Biol Chem. 2006; 281(51): 39681–92. PubMed Abstract \| Publisher Full Text\n\nLin Y, Rao J, Zha XL, et al.: Angiopoietin-like 3 induces podocyte F-actin rearrangement through integrin α(V)β₃/FAK/PI3K pathway-mediated Rac1 activation. Biomed Res Int. 2013; 2013: 135608. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFiorina P, Vergani A, Bassi R, et al.: Role of podocyte B7-1 in diabetic nephropathy. J Am Soc Nephrol. 2014; 25(7): 1415–29. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nIglesias-de la Cruz MC, Ziyadeh FN, Isono M, et al.: Effects of high glucose and TGF-beta1 on the expression of collagen IV and vascular endothelial growth factor in mouse podocytes. Kidney Int. 2002; 62(3): 901–13. PubMed Abstract \| Publisher Full Text\n\nHan SY, Kang YS, Jee YH, et al.: High glucose and angiotensin II increase beta1 integrin and integrin-linked kinase synthesis in cultured mouse podocytes. Cell Tissue Res. 2006; 323(2): 321–32. PubMed Abstract \| Publisher Full Text\n\nHa TS: High glucose and advanced glycosylated end-products affect the expression of alpha-actinin-4 in glomerular epithelial cells. Nephrology (Carlton). 2006; 11(5): 435–41. PubMed Abstract \| Publisher Full Text\n\nKim NH, Rincon-Choles H, Bhandari B, et al.: Redox dependence of glomerular epithelial cell hypertrophy in response to glucose. Am J Physiol Renal Physiol. 2006; 290(3): F741–51. PubMed Abstract \| Publisher Full Text\n\nDe Petris L, Hruska KA, Chiechio S, et al.: Bone morphogenetic protein-7 delays podocyte injury due to high glucose. Nephrol Dial Transplant. 2007; 22(12): 3442–50. PubMed Abstract \| Publisher Full Text\n\nWei C, El Hindi S, Li J, et al.: Circulating urokinase receptor as a cause of focal segmental glomerulosclerosis. Nat Med. 2011; 17(8): 952–60. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nKoukouritaki SB, Vardaki EA, Papakonstanti EA, et al.: TNF-alpha induces actin cytoskeleton reorganization in glomerular epithelial cells involving tyrosine phosphorylation of paxillin and focal adhesion kinase. Mol Med. 1999; 5(6): 382–92. PubMed Abstract \| Free Full Text\n\nSaito Y, Okamura M, Nakajima S, et al.: Suppression of nephrin expression by TNF-alpha via interfering with the cAMP-retinoic acid receptor pathway. Am J Physiol Renal Physiol. 2010; 298(6): F1436–44. PubMed Abstract \| Publisher Full Text\n\nBitzan M, Babayeva S, Vasudevan A, et al.: TNFα pathway blockade ameliorates toxic effects of FSGS plasma on podocyte cytoskeleton and β3 integrin activation. Pediatr Nephrol. 2012; 27(12): 2217–26. PubMed Abstract \| Publisher Full Text\n\nTian D, Jacobo SM, Billing D, et al.: Antagonistic regulation of actin dynamics and cell motility by TRPC5 and TRPC6 channels. Sci Signal. 2010; 3(145): ra77. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nYoshida S, Nagase M, Shibata S, et al.: Podocyte injury induced by albumin overload in vivo and in vitro: involvement of TGF-beta and p38 MAPK. Nephron Exp Nephrol. 2008; 108(3): e57–68. PubMed Abstract \| Publisher Full Text\n\nHe F, Chen S, Wang H, et al.: Regulation of CD2-associated protein influences podocyte endoplasmic reticulum stress-mediated apoptosis induced by albumin overload. Gene. 2011; 484(1–2): 18–25. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKampe K, Sieber J, Orellana JM, et al.: Susceptibility of podocytes to palmitic acid is regulated by fatty acid oxidation and inversely depends on acetyl-CoA carboxylases 1 and 2. Am J Physiol Renal Physiol. 2014; 306(4): F401–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSusztak K, Raff AC, Schiffer M, et al.: Glucose-induced reactive oxygen species cause apoptosis of podocytes and podocyte depletion at the onset of diabetic nephropathy. Diabetes. 2006; 55(1): 225–33. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nPeters I, Tossidou I, Achenbach J, et al.: IGF-binding protein-3 modulates TGF-beta/BMP-signaling in glomerular podocytes. J Am Soc Nephrol. 2006; 17(6): 1644–56. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nBussolati B, Deregibus MC, Fonsato V, et al.: Statins prevent oxidized LDL-induced injury of glomerular podocytes by activating the phosphatidylinositol 3-kinase/AKT-signaling pathway. J Am Soc Nephrol. 2005; 16(7): 1936–47. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nSchiffer M, Mundel P, Shaw AS, et al.: A novel role for the adaptor molecule CD2-associated protein in transforming growth factor-beta-induced apoptosis. J Biol Chem. 2004; 279(35): 37004–12. PubMed Abstract \| Publisher Full Text\n\nWada T, Pippin JW, Terada Y, et al.: The cyclin-dependent kinase inhibitor p21 is required for TGF-beta1-induced podocyte apoptosis. Kidney Int. 2005; 68(4): 1618–29. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nWu DT, Bitzer M, Ju W, et al.: TGF-beta concentration specifies differential signaling profiles of growth arrest/differentiation and apoptosis in podocytes. J Am Soc Nephrol. 2005; 16(11): 3211–21. PubMed Abstract \| Publisher Full Text\n\nJung KY, Chen K, Kretzler M, et al.: TGF-beta1 regulates the PINCH-1-integrin-linked kinase-alpha-parvin complex in glomerular cells. J Am Soc Nephrol. 2007; 18(1): 66–73. PubMed Abstract \| Publisher Full Text\n\nYadav A, Vallabu S, Arora S, et al.: ANG II promotes autophagy in podocytes. Am J Physiol Cell Physiol. 2010; 299(2): C488–96. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMa T, Zhu J, Chen X, et al.: High glucose induces autophagy in podocytes. Exp Cell Res. 2013; 319(6): 779–89. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu Y: New insights into epithelial-mesenchymal transition in kidney fibrosis. J Am Soc Nephrol. 2010; 21(2): 212–22. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi Y, Kang YS, Dai C, et al.: Epithelial-to-mesenchymal transition is a potential pathway leading to podocyte dysfunction and proteinuria. Am J Pathol. 2008; 172(2): 299–308. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHusain M, Gusella GL, Klotman ME, et al.: HIV-1 Nef induces proliferation and anchorage-independent growth in podocytes. J Am Soc Nephrol. 2002; 13(7): 1806–15. PubMed Abstract \| Publisher Full Text\n\nSunamoto M, Husain M, He JC, et al.: Critical role for Nef in HIV-1-induced podocyte dedifferentiation. Kidney Int. 2003; 64(5): 1695–701. PubMed Abstract \| Publisher Full Text\n\nBruggeman LA, Drawz PE, Kahoud N, et al.: TNFR2 interposes the proliferative and NF-κB-mediated inflammatory response by podocytes to TNF-α. Lab Invest. 2011; 91(3): 413–25. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nClement LC, Macé C, Avila-Casado C, et al.: Circulating angiopoietin-like 4 links proteinuria with hypertriglyceridemia in nephrotic syndrome. Nat Med. 2014; 20(1): 37–46. 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PubMed Abstract \| Publisher Full Text\n\nFoster RR, Saleem MA, Mathieson PW, et al.: Vascular endothelial growth factor and nephrin interact and reduce apoptosis in human podocytes. Am J Physiol Renal Physiol. 2005; 288(1): F48–57. PubMed Abstract \| Publisher Full Text\n\nMüller-Deile J, Worthmann K, Saleem M, et al.: The balance of autocrine VEGF-A and VEGF-C determines podocyte survival. Am J Physiol Renal Physiol. 2009; 297(6): F1656–67. PubMed Abstract \| Publisher Full Text\n\nFoster RR, Satchell SC, Seckley J, et al.: VEGF-C promotes survival in podocytes. Am J Physiol Renal Physiol. 2006; 291(1): F196–207. PubMed Abstract \| Publisher Full Text\n\nBarisoni L, Schnaper HW, Kopp JB: A proposed taxonomy for the podocytopathies: a reassessment of the primary nephrotic diseases. Clin J Am Soc Nephrol. 2007; 2(3): 529–42. PubMed Abstract \| Publisher Full Text\n\nChiang C, Inagi R: Glomerular diseases: genetic causes and future therapeutics. Nat Rev Nephrol. 2010; 6(9): 539–54. 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PubMed Abstract \| Publisher Full Text\n\nMedapalli RK, He JC, Klotman PE: HIV-associated nephropathy: pathogenesis. Curr Opin Nephrol Hypertens. 2011; 20(3): 306–11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLaurinavicius A, Rennke HG: Collapsing glomerulopathy--a new pattern of renal injury. Semin Diagn Pathol. 2002; 19(3): 106–15. PubMed Abstract\n\nBarri YM, Munshi NC, Sukumalchantra S, et al.: Podocyte injury associated glomerulopathies induced by pamidronate. Kidney Int. 2004; 65(2): 634–41. PubMed Abstract \| Publisher Full Text\n\nAlbaqumi M, Soos TJ, Barisoni L, et al.: Collapsing glomerulopathy. J Am Soc Nephrol. 2006; 17(10): 2854–63. PubMed Abstract \| Publisher Full Text\n\nDetwiler RK, Falk RJ, Hogan SL, et al.: Collapsing glomerulopathy: a clinically and pathologically distinct variant of focal segmental glomerulosclerosis. Kidney Int. 1994; 45(5): 1416–24. 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PubMed Abstract \| Publisher Full Text\n\nZhou X, Hurst RD, Templeton D, et al.: High glucose alters actin assembly in glomerular mesangial and epithelial cells. Lab Invest. 1995; 73(3): 372–83. PubMed Abstract\n\nChen HM, Liu ZH, Zeng CH, et al.: Podocyte lesions in patients with obesity-related glomerulopathy. Am J Kidney Dis. 2006; 48(5): 772–9. PubMed Abstract \| Publisher Full Text\n\nGriffin SV, Petermann AT, Durvasula RV, et al.: Podocyte proliferation and differentiation in glomerular disease: role of cell-cycle regulatory proteins. Nephrol Dial Transplant. 2003; 18(Suppl 6): vi8–13. PubMed Abstract \| Publisher Full Text\n\nKriz W: Podocyte is the major culprit accounting for the progression of chronic renal disease. Microsc Res Tech. 2002; 57(4): 189–95. PubMed Abstract \| Publisher Full Text\n\nKikuchi M, Wickman L, Hodgin JB, et al.: Podometrics as a Potential Clinical Tool for Glomerular Disease Management. Semin Nephrol. 2015; 35(3): 245–55. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nReiser J, Gupta V, Kistler AD: Toward the development of podocyte-specific drugs. Kidney Int. 2010; 77(8): 662–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChugh SS, Macé C, Clement LC, et al.: Angiopoietin-like 4 based therapeutics for proteinuria and kidney disease. Front Pharmacol. 2014; 5: 23. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFornoni A, Sageshima J, Wei C, et al.: Rituximab targets podocytes in recurrent focal segmental glomerulosclerosis. Sci Transl Med. 2011; 3(85): 85ra46. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nYu CC, Fornoni A, Weins A, et al.: Abatacept in B7-1-positive proteinuric kidney disease. N Engl J Med. 2013; 369(25): 2416–23. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nBenigni A, Gagliardini E, Remuzzi G: Abatacept in B7-1-positive proteinuric kidney disease. N Engl J Med. 2014; 370(13): 1261–3. PubMed Abstract \| Publisher Full Text\n\nAlachkar N, Carter-Monroe N, Reiser J: Abatacept in B7-1-positive proteinuric kidney disease. N Engl J Med. 2014; 370(13): 1263–4. PubMed Abstract \| Publisher Full Text\n\nSalant DJ: Podocyte Expression of B7-1/CD80: Is it a Reliable Biomarker for the Treatment of Proteinuric Kidney Diseases with Abatacept? J Am Soc Nephrol. 2015; pii: ASN.2015080947. PubMed Abstract \| Publisher Full Text\n\nSchiffer M, Teng B, Gu C, et al.: Pharmacological targeting of actin-dependent dynamin oligomerization ameliorates chronic kidney disease in diverse animal models. Nat Med. 2015; 21(6): 601–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee HW, Khan SQ, Faridi MH, et al.: A Podocyte-Based Automated Screening Assay Identifies Protective Small Molecules. J Am Soc Nephrol. 2015; 26(11): 2741–52. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "12154", "date": "28 Jan 2016", "name": "Sumant Chugh", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12155", "date": "28 Jan 2016", "name": "Farhad Danesh", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-114
https://f1000research.com/articles/5-113/v1	28 Jan 16	{ "type": "Review", "title": "Advances in the endoscopic diagnosis and treatment of Barrett’s neoplasia", "authors": [ "Fergus J.Q. Chedgy", "Kesavan Kandiah", "Sreedhari Thayalasekaran", "Sharmila Subramaniam", "Pradeep Bhandari", "Fergus J.Q. Chedgy", "Kesavan Kandiah", "Sreedhari Thayalasekaran", "Sharmila Subramaniam" ], "abstract": "Barrett’s oesophagus is a well-recognised precursor of oesophageal adenocarcinoma. The incidence of oesophageal adenocarcinoma is continuing to rise in the Western world with dismal survival rates. In recent years, efforts have been made to diagnose Barrett’s earlier and improve surveillance techniques in order to pick up cancerous changes earlier. Recent advances in endoscopic therapy for early Barrett’s cancers have shifted the paradigm away from oesophagectomy and have yielded excellent results.", "keywords": [ "Endoscopy", "Barrett’s neoplasia", "Barrett’s oesophagus", "oesophageal adenocarcinoma" ], "content": "Introduction\n\nBarrett’s oesophagus (BO) is defined as the replacement of the normal distal oesophageal squamous epithelium with metaplastic columnar epithelium1. This metaplastic epithelium accumulates genetic changes that over time can progress to dysplasia and cancer2. Screening for BO with endoscopy remains controversial because of poor uptake and lack of cost-effectiveness. A number of alternatives have been proposed to remedy the situation: cytosponge, transnasal endoscopy (TNE) and oesophageal capsule endoscopy (OCE). Surveillance of BO has been demonstrated to diagnose oesophageal cancers earlier and provide a better prognosis3. Surveillance has been improved by advances in endoscope technology and techniques, including high-definition endoscopy and chromoendoscopy. The advantage of detecting oesophageal neoplasia at an early stage is that it can be successfully treated endoscopically without resorting to oesophagectomy.\n\n\nScreening for Barrett’s oesophagus\n\nThere is a lack of high-quality evidence supporting the use of conventional endoscopy in population-based screening for BO. Ten percent of patients with gastro-oesophageal reflux disease (GORD) have BO4. However, even if screening were performed for every adult with GORD, 40% of oesophageal adenocarcinomas would still be missed5. This has prompted research to identify less invasive and more cost-effective and acceptable methods to screen for BO, such as cytosponge, TNE and OCE.\n\nThe cytosponge is a sponge contained within a capsule that is attached to a string. The capsule is ingested with water and dissolves in the stomach after 3 to 5 minutes. The string then is pulled to retrieve the sponge and cells collected from the oesophagus6,7. The cells are analysed with the biomarker trefoil factor 3 to make a diagnosis of BO.\n\nAn initial study of the cytosponge6 demonstrated a 3% pickup rate of BO in a primary care setting with a majority of patients (82%) reporting low levels of anxiety, making it a potential tool for mass screening. More recently, Fitzgerald et al. followed up on their work with the cytosponge in a large case-control study8. In total, 1,110 patients were recruited: 463 patients with symptoms of dyspepsia and reflux and 647 patients with a prior diagnosis of BO underwent gastroscopy following cytosponge examination; 93.9% of patients had a successful cytosponge examination. Overall, cytosponge sensitivity for detecting BO was 79.9%, increasing to 87.2% in patients with more than 3 cm of BO. The specificity for diagnosing BO was 92.4%. Further trials on the cytosponge device are ongoing but these data suggest that it is an acceptable and accurate device.\n\nA recent randomised controlled trial compared the use of unsedated TNE versus sedated gastroscopy for BO screening. Two hundred and nine patients were recruited to standard gastroscopy (surveillance oesophago-gastro-duodenoscopy, or sOGD), unsedated TNE in a mobile research van (muTNE) or a hospital outpatient endoscopy suite (huTNE). Uptake was greater in the unsedated TNE group: 47.5% for muTNE and 45.7% for huTNE versus 40.7% for standard gastroscopy. Complete evaluation of the oesophagus was similar between the groups: 99% muTNE, 96% huTNE and 100% sOGD9.\n\nChak et al.10 examined the acceptability of TNE versus OCE in a randomised controlled trial. They found that uptake for screening examination was low: 15.2% of patients (n = 1,210). Effectiveness of screening for the detection of BO was similar for both technologies. A meta-analysis11 of studies investigating OCE as a screening modality for BO in patients with reflux symptoms demonstrated an overall sensitivity of 77% and a specificity of 86%.\n\nAlthough technology is advancing in the field of BO screening, there are insufficient data about its cost-effectiveness. These novel approaches appear to be acceptable to patients but more data are required to target whom and when to offer screening.\n\n\nSurveillance for Barrett’s oesophagus\n\nIn British guidelines the presence of columnar lined oesophagus alone is considered acceptable for a diagnosis of Barrett’s1. American guidelines differ in that they require histological confirmation of intestinal metaplasia to confirm a diagnosis of BO12. Presence of intestinal metaplasia poses a greater risk of neoplastic transformation, and intestinal metaplasia is generally present in longer segments of Barrett’s. The current British guidelines recommend that Barrett’s segments of more than 3 cm have a surveillance OGD every 2 to 3 years and segments of less than 3 cm with the presence of intestinal metaplasia have surveillance OGD every 3 to 5 years. Likewise, the American Society for Gastrointestinal Endoscopy recommends surveillance OGD every 3 to 5 years.\n\n\nEndoscopic diagnosis\n\nThe annual rate of transformation into oesophageal adenocarcinoma (OAC) in patients with non-dysplastic BO is estimated to be between 0.07% and 0.82%13–15. However, the annual rate of progression from low-grade dysplasia to high-grade dysplasia (HGD) or OAC is as high as 6.5%16–18 and from HGD to OAC is 12% to 40%19,20. Dysplasia in Barrett’s is often flat, patchy and difficult to detect. British Society of Gastroenterology (BSG) guidelines1 recommend the Seattle biopsy protocol, which entails four-quadrant random biopsies every 2 cm in addition to targeted biopsies on macroscopically visible lesions. This surveillance method has had a poor uptake amongst endoscopists as it is time-consuming, labour-intensive, and prone to sampling error21,22.\n\nSimple techniques such as mucolytic agents and increased inspection times can be used in order to improve visualisation of Barrett’s mucosa during surveillance. N-acetylcysteine is a mucolytic agent that can be used at a concentration of 4% to 10% to dissolve excess mucus and bubbles. Basford et al.23 recently reported on a randomised controlled trial (n = 126 patients) comparing a combination of simeticone and N-acetylcysteine (NAC) (group A) as a pre-drink prior to gastroscopy with water alone (group B) and no pre-drink. They reported significant improvement in mucosal visibility with simeticone and NAC as compared with just water or no pre-drink. This pre-drink was also reported to reduce the number of additional flushes to achieve satisfactory views. There is also evidence, in a similar way to colonoscopy, that the longer the duration spent assessing BO, the greater the detection rate for neoplasia24. The study suggests spending at least 1 minute per centimetre of Barrett’s. However, this study was performed in a high-risk tertiary referral population in whom the index of suspicion of neoplasia was high, and therefore may not apply to the routine surveillance population.\n\nWith the advent of charge-coupled device (CCD) chips, high-definition white light (HDWL) endoscopes are able to capture and display high-definition images with pixel densities of more than 10 million pixels, making standard definition (pixel density of 100,000 to 400,000) endoscopes obsolete25.\n\nThe sensitivity and specificity of HDWL endoscopy in detecting Barrett’s neoplasia are 40%–64% and 98%–100%, respectively26,27. BSG and American Gastroenterological Association (AGA) guidelines recommend the use of high-resolution endoscopes for Barrett’s surveillance1,28.\n\nThe enhancement of mucosal surface and vascular patterns using optical and digital filter technologies has added to the arsenal of the advanced endoscopist in the quest to improve dysplasia detection. Narrow band imaging (NBI) (Olympus, Tokyo, Japan) and blue laser imaging (BLI) (Fujifilm, Tokyo, Japan) use a filter located in front of the light source. This technology filters white light and limits the wavelength of the light projected to 415 to 540 nm29. When projected onto a mucosal surface, this ‘narrow band’ of light appears blue and green. The blue light penetrates the superficial layer of the mucosa, thereby enhancing the view of superficial capillaries and the crypt patterns in the mucosal surface. In contrast, technologies such as i-Scan (Pentax, Tokyo, Japan) and Fujinon intelligent chromoendoscopy (FICE) (Fujinon, Tokyo, Japan) employ complex proprietary algorithms to digitally reproduce a narrow-spectrum image at the push of a button on the endoscope. BLI30 is a new technology with white and blue lasers that produce narrow-band light.\n\nNarrow band imaging. NBI is the most studied optical imaging technology thus far and has a sensitivity and specificity of 47%–100% and 72%–100%, respectively, for detecting Barrett’s neoplasia27,31–34. NBI selectively enhances mucosal vascular patterns by narrowing the spectrum of light, reducing the amount of red light in the displayed image whilst narrowing the spectrum of blue and green, making blood vessels appear dark against the background mucosa35.\n\nA majority of these studies were conducted in tertiary referral centres by expert endoscopists evaluating an enriched population with a high index of suspicion of neoplasia. The technology has yet to be validated in non-expert hands or in a surveillance population. Therefore, we would suggest that training in the use of this technology and data in a surveillance population be required prior to adoption in routine clinical practice.\n\ni-Scan. i-Scan uses post-processor technology that reconstructs the image transmitted from the endoscope by using a computer-based algorithm which is able to accentuate both surface patterns and vasculature35. A randomised control trial found that the yields of acetic acid-guided versus i-Scan-guided biopsies in detecting specialised columnar epithelium were comparable36. Verna et al. found that dysplasia detection rate using this technology was inferior to that using the standard four-quadrant biopsy technique37. However, these studies were poorly designed and have small sample sizes. More robust studies on the utility of i-Scan in the detection of dysplasia in BO are required.\n\nFujinon intelligent chromoendoscopy. FICE uses a CCD in the endoscope to capture spectral reflectance data. A matrix processing circuit found in the video processor then receives the data. The reflectance spectra of corresponding pixels that make up the conventional image are mathematically estimated. From this information, a single-wavelength virtual image is reconstructed. Three such single-wavelength images can be selected and assigned to the red, green and blue monitor inputs to display a composite colour-enhanced multiband image in real time. This can be used like NBI to remove data from the red part of the waveband and narrow the green and blue spectra35.\n\nThere is a paucity of research evaluating the utility of FICE in detection of dysplasia in Barrett’s. The sole published study to date is a prospective pilot study carried out in a tertiary centre. It was found that the dysplasia detection rate of FICE, when used in conjunction with acetic acid, is 86%38.\n\nAutofluorescence imaging. Autofluorescence imaging (AFI) is based on a principle that a specific light wavelength can cause fluorescence of endogenous biomolecules such as collagen, nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), and porphyrins. These molecules can accumulate in dysplastic oesophageal mucosa39. A randomised cross-over multi-centre trial on an enriched population found a marginal gain of AFI over quadrantic biopsies which did not reach statistical significance40. Endoscopic trimodal imaging (ETMI) systems which integrate AFI with HDWL and NBI have not been shown to be superior to standard resolution white light endoscopes41,42. With a lack of evidence of its efficacy and high false-positive rates, the use of AFI and ETMI at present remains in the domain of endoscopic research39–41.\n\nMethylene blue chromoendoscopy. Three randomised cross-over trials found that the diagnostic accuracy of methylene blue 0.5%-directed biopsies is higher than random 2 cm quadrantic biopsies43–45. However, a meta-analysis which included data from six trials found that overall methylene blue chromoendoscopy was not superior to random biopsies in the detection of specialised intestinal metaplasia or dysplasia46.\n\nAcetic acid chromoendoscopy. Acetic acid is a weak acid that causes acetowhitening of the oesophageal mucosa. Over a period of seconds to minutes, dysplastic tissue will start to lose the acetowhitening effect before the surrounding non-dysplastic Barrett’s tissue. Differential loss of acetowhitening highlights the neoplastic focus as a red spot on a white background (Figure 1). This is an extremely promising technique with high sensitivity, universal applicability and negligible cost.\n\nTwo large cohort studies have demonstrated effectiveness of acetic acid used at concentrations of 2.5% and 1.5%, respectively47,48, in the detection of dysplasia within Barrett’s in high-risk populations. The two reported similar results, with sensitivities for dysplasia between 90% and 95% and specificities between 75% and 85%. A further study using 2.5% acetic acid found that the number of biopsies needed to detect neoplasia could be significantly reduced if acetic acid targeted biopsies were used in place of mapping biopsies, thereby reducing pathology-related costs by 97%49. Tholoor et al.50 reported the use of 2.5% acetic acid in a surveillance population and were able to demonstrate a threefold increase in neoplasia detection as compared with conventional protocol-guided biopsies. However, this was a non-randomised trial and an ongoing randomised trial, the ABBA study51, will answer this question soon. The ABBA study is a randomised, crossover, tandem endoscopy study comparing standard quadrantic biopsy protocol versus acetic acid targeted biopsies, in a Barrett’s surveillance population.\n\nCross-sectional optical imaging. Optical coherence tomography (OCT) and confocal laser endomicroscopy (CLE) are emerging technologies that are able to obtain micro-anatomical images of the oesophageal mucosa. OCT technology measures the difference between the backscatter of near-infrared low-coherence light below the tissue surface and a reference beam52. Using this information, it is able to reconstruct the microanatomy of the superficial mucosal layer53. CLE, on the other hand, involves the integration of a confocal laser microscope in the distal tip of a conventional video endoscope54. Although both technologies have sensitivities of 68% to 86% and specificities of 73% to 83%55,56, the setup costs are high and special training is required for image interpretation. This currently limits its use in routine clinical practice. Table 1 summarises the performance of current imaging technologies in the diagnosis of Barrett’s neoplasia.\n\n\nEndotherapy\n\nUntil recently, oesophagectomy was regarded as the gold standard treatment for patients with HGD or early oesophageal cancer57. Even in expert hands, oesophagectomy carries significant morbidity and mortality: 30% to 50% and 2% to 5%, respectively58–60. Not only are surgical risks high but the patient population with this condition have co-morbidities that often preclude surgical intervention. In the last 15 years, endoscopic therapy has become an established treatment of HGD and intramucosal adenocarcinoma (T1a) as the risk of lymph node metastases is very low1.\n\nExperience of endoscopic resection (ER) for early oesophageal adenocarcinoma began in the early 1990s in Asia; since then, techniques have significantly progressed. Initial experience came from the strip biopsy technique, which was further refined by the suck-and-cut and multi-band ligator techniques (Figure 2)61. Ell et al.62 reported the first (n = 64) series of ER for early Barrett’s cancer, demonstrating a 97% remission rate. Despite a short follow-up period (mean of 12 months), there was a significant rate of recurrence of 14%.\n\n(a) An area of Barrett’s high-grade dysplasia. (b) The same area demonstrating acetowhitening effect. (c) The same lesion as viewed down a multi-band ligator. (d) Pseudopolyp created by the band ligator. (e) Resection defect following endoscopic mucosal resection.\n\nIn the early years, a high rate of metachronous and recurrent lesions hampered the apparent technical success of ER with reported recurrence rates of up to 35%52. This led to a refinement of techniques incorporating both endoscopic mucosal resection (EMR) and ablative therapies (discussed below). A randomised trial63 (The APE Study) comparing EMR + argon plasma coagulation (APC) ablation versus EMR + observation demonstrated a significantly reduced risk of metachronous lesions in the ablation arm, 3% versus 37%, such that ablation following ER is now standard treatment.\n\nPech et al.52 reported their outcome data of 1,000 patients with early Barrett’s cancer treated by ER, demonstrating a long-term complete remission rate of 93.8%. There was a 14.5% recurrence rate, and a majority of recurrences were treated endoscopically. Their serious complication rate was 1.5%, and no mortality was reported.\n\n\nAblative therapies\n\nGiven the recognition that ablation following ER offers optimum outcomes in terms of neoplasia eradication, it now forms an essential component of the treatment pathway of early Barrett’s cancer. The two main techniques are APC and radiofrequency ablation (RFA). The findings of the APE study63 are described above. A recent meta-analysis64 of RFA has shown intestinal metaplasia eradication rates of 78%, dysplasia eradication rates of 91%, and cancer progression rates of 0.2% to 0.5% with an oesophageal stricture rate of 5%. Data from the UK RFA registry65 report similar rates of success with a complete dysplasia remission rate of 92% and a stricture rate of 6.2%.\n\nThere are currently no data comparing outcomes of APC and RFA to determine which is more effective. However, the Barrett’s Intervention for Dysplasia by Endoscopy (BRIDE) study aims to answer this question. Whilst RFA is an extremely effective treatment, its costs remain high (up to £1,800 per catheter), and EMR followed by intensive acetic acid surveillance has been shown to produce outcomes similar to those of the UK RFA registry at a significantly cheaper cost66.\n\nEarly experience with cryotherapy shows promising results with dysplasia eradication rates of up to 97% in patients with short-segment BO67. This technology, however, remains firmly in the research domain until further data are available.\n\nThe main limitation of the EMR technique is that en bloc resection is possible only for lesions of less than 15 mm; lesions larger than this require piecemeal resection, making adequate histological assessment difficult. Figure 3 demonstrates the steps involved in the endoscopic submucosal dissection (ESD) technique. Experience with ESD in Japanese studies of early oesophageal squamous cell cancer has demonstrated improved outcomes over EMR. To date, three European studies have reported outcomes of ESD for neoplastic Barrett’s. Neuhaus et al.68 reported on 30 patients with early neoplastic lesions up to 30 mm: en bloc resection was achieved in 90%, the complete neoplasia eradication rate was 96.4%, and there were no reported complications. More recently, Chevaux et al.69 reported on their outcomes of 75 consecutive patients; ESD was performed on lesions of more than 15 mm, achieving an en bloc resection rate of 90% and a neoplasia eradication rate of 92%, and oesophageal strictures developed in 60% of patients. Probst et al.70 reported on 87 patients with early oesophageal adenocarcinoma achieving a 95.4% en bloc resection rate with a recurrence rate of 2.4%; 11.7% of patients had stricturing, and no perforations were reported. Disappointingly, however, these studies have reported low R0 resection rates (38.5% to 48.5%).\n\n(a) pT1a/M3 intramucosal cancer arising in Barrett’s oesophagus. (b) The same lesion following acetic acid. Note the differential early loss of acetowhitening. (c) Edges of the lesion marked with endoscopic submucosal dissection (ESD) knife under virtual chromoendoscopy. (d) Submucosal injection. (e) Mucosal incision with ESD knife. (f) Resection base following ESD. (g) Resection specimen.\n\nESD appears to be a promising addition to current treatment techniques, especially for larger lesions. Our own data on 51 ESDs, on selected patients with Barrett’s cancer, have demonstrated an R0 resection rate for cancer of 88%, a recurrence rate of 3% and no complications71. A recently reported randomised controlled trial of EMR (n = 20) versus ESD (n = 31) demonstrated superior en bloc, R0 and curative resection rates for ESD; however, there was no difference in clinical outcome for either technique72. In our experience, large nodular lesions have a high risk of containing cancer and therefore we believe they should be removed in an en bloc fashion. Early European data have shown the feasibility and safety of ESD in Barrett’s neoplasia but have not proven its superiority over EMR. This has to be addressed in a well-designed study for a select group of patients to identify the exact role of ESD in Barrett’s neoplasia.\n\n\nConclusions\n\nAt present, there is insufficient evidence to advocate population-based screening, and novel techniques, though acceptable to patients, have yet to be proven cost-effective. Inevitably advances in endoscope technology will improve dysplasia detection, but we believe formalised training programmes are required to extrapolate trial evidence from expert endoscopists into everyday practice. Outcomes of endotherapy for early Barrett’s neoplasia are excellent and should be considered first-line treatment in this group. Increasing experience of ESD in the West will enable en bloc resection of larger and more advanced lesions with good outcomes, but further trial data are required to clarify who benefits most from this technique.\n\n\nAbbreviations\n\nAFI, autofluorescence imaging; APC, argon plasma coagulation; BLI, blue laser imaging; BO, Barrett’s oesophagus; BSG, British Society of Gastroenterology; CCD, charge-coupled device; CLE, confocal laser endomicroscopy; EMR, endoscopic mucosal resection; ER, endoscopic resection; ESD, endoscopic submucosal dissection; ETMI, endoscopic trimodal imaging; FICE, Fujinon intelligent chromoendoscopy; GORD, gastro-oesophageal reflux disease; HDWL, high-definition white light; HGD, high-grade dysplasia; huTNE, unsedated transnasal endoscopy in a hospital outpatient endoscopy suite; muTNE, unsedated transnasal endoscopy in a mobile research van; NAC, N-acetylcysteine; NBI, narrow band imaging; OAC, oesophageal adenocarcinoma; OCE, oesophageal capsule endoscopy; OCT, optical coherence tomography; RFA, radiofrequency ablation; sOGD, surveillance oesophago-gastro-duodenoscopy; TNE, transnasal endoscopy.", "appendix": "Competing interests\n\n\n\nThe authors declare that they 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\nFitzgerald RC, di Pietro M, Ragunath K, et al.: British Society of Gastroenterology guidelines on the diagnosis and management of Barrett's oesophagus. 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BMJ. 2010; 341: c4372. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nLao-Sirieix P, Boussioutas A, Kadri SR, et al.: Non-endoscopic screening biomarkers for Barrett's oesophagus: from microarray analysis to the clinic. Gut. 2009; 58(11): 1451–9. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nRoss-Innes CS, Debiram-Beecham I, O'Donovan M, et al.: Evaluation of a minimally invasive cell sampling device coupled with assessment of trefoil factor 3 expression for diagnosing Barrett's esophagus: a multi-center case-control study. PLoS Med. 2015; 12(1): e1001780. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSami SS, Dunagan KT, Johnson ML, et al.: A randomized comparative effectiveness trial of novel endoscopic techniques and approaches for Barrett's esophagus screening in the community. Am J Gastroenterol. 2015; 110(1): 148–58. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nChak A, Alashkar BM, Isenberg GA, et al.: Comparative acceptability of transnasal esophagoscopy and esophageal capsule esophagoscopy: a randomized, controlled trial in veterans. Gastrointest Endosc. 2014; 80(5): 774–82. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nBhardwaj A, Hollenbeak CS, Pooran N, et al.: A meta-analysis of the diagnostic accuracy of esophageal capsule endoscopy for Barrett's esophagus in patients with gastroesophageal reflux disease. Am J Gastroenterol. 2009; 104(6): 1533–9. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nASGE Standards of Practice Committee, Evans JA, Early DS, et al.: The role of endoscopy in Barrett's esophagus and other premalignant conditions of the esophagus. Gastrointest Endosc. 2012; 76(6): 1087–94. PubMed Abstract \| Publisher Full Text\n\nde Jonge PJ, van Blankenstein M, Grady WM, et al.: Barrett's oesophagus: epidemiology, cancer risk and implications for management. Gut. 2014; 63(1): 191–202. PubMed Abstract \| Publisher Full Text\n\nHvid-Jensen F, Pedersen L, Drewes AM, et al.: Incidence of adenocarcinoma among patients with Barrett's esophagus. N Engl J Med. 2011; 365(15): 1375–83. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nYousef F, Cardwell C, Cantwell MM, et al.: The incidence of esophageal cancer and high-grade dysplasia in Barrett's esophagus: a systematic review and meta-analysis. Am J Epidemiol. 2008; 168(1): 237–49. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nSharma P, Falk GW, Weston AP, et al.: Dysplasia and cancer in a large multicenter cohort of patients with Barrett's esophagus. Clin Gastroenterol Hepatol. 2006; 4(5): 566–72. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nPicardo SL, O'Brien MP, Feighery R, et al.: A Barrett's esophagus registry of over 1000 patients from a specialist center highlights greater risk of progression than population-based registries and high risk of low grade dysplasia. Dis Esophagus. 2015; 28(2): 121–6. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nSingh S, Manickam P, Amin AV, et al.: Incidence of esophageal adenocarcinoma in Barrett's esophagus with low-grade dysplasia: a systematic review and meta-analysis. Gastrointest Endosc. 2014; 79(6): 897–909.e4; quiz 983.e1, 983.e3. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nKonda VJ, Ross AS, Ferguson MK, et al.: Is the risk of concomitant invasive esophageal cancer in high-grade dysplasia in Barrett's esophagus overestimated? Clin Gastroenterol Hepatol. 2008; 6(2): 159–64. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nHeitmiller RF, Redmond M, Hamilton SR: Barrett's esophagus with high-grade dysplasia. An indication for prophylactic esophagectomy. Ann Surg. 1996; 224(1): 66–71. PubMed Abstract \| Free Full Text\n\nPeters FP, Curvers WL, Rosmolen WD, et al.: Surveillance history of endoscopically treated patients with early Barrett's neoplasia: nonadherence to the Seattle biopsy protocol leads to sampling error. Dis Esophagus. 2008; 21(6): 475–9. PubMed Abstract \| Publisher Full Text\n\nKariv R, Plesec TP, Goldblum JR, et al.: The Seattle protocol does not more reliably predict the detection of cancer at the time of esophagectomy than a less intensive surveillance protocol. Clin Gastroenterol Hepatol. 2009; 7(6): 653–8; quiz 606. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nBasford P, Brown J, Gadeke L, et al.: NICEVIS - results of a randomised controlled trial of simeticone and N-acetylcysteine as a pre-procedure drink to improve mucosal visibility during diagnostic gastroscopy. UEGW. 2015. Reference Source\n\nGupta N, Gaddam S, Wani SB, et al.: Longer inspection time is associated with increased detection of high-grade dysplasia and esophageal adenocarcinoma in Barrett's esophagus. Gastrointest Endosc. 2012; 76(3): 531–8. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nASGE Technology Committee, Kwon RS, Adler DG, et al.: High-resolution and high-magnification endoscopes. Gastrointest Endosc. 2009; 69(3 Pt 1): 399–407. PubMed Abstract \| Publisher Full Text\n\nCanto MI, Anandasabapathy S, Brugge W, et al.: In vivo endomicroscopy improves detection of Barrett's esophagus-related neoplasia: a multicenter international randomized controlled trial (with video). Gastrointest Endosc. 2014; 79(2): 211–21. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nSharma P, Hawes RH, Bansal A, et al.: Standard endoscopy with random biopsies versus narrow band imaging targeted biopsies in Barrett's oesophagus: a prospective, international, randomised controlled trial. Gut. 2013; 62(1): 15–21. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nSpechler SJ, Sharma P, Souza RF, et al.: American Gastroenterological Association technical review on the management of Barrett's esophagus. Gastroenterology. 2011; 140(3): e18–52; quiz e13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGono K, Obi T, Yamaguchi M, et al.: Appearance of enhanced tissue features in narrow-band endoscopic imaging. J Biomed Opt. 2004; 9(3): 568–77. PubMed Abstract \| Publisher Full Text\n\nYoshida N, Hisabe T, Hirose R, et al.: Improvement in the visibility of colorectal polyps by using blue laser imaging (with video). Gastrointest Endosc. 2015; 82(3): 542–9. PubMed Abstract \| Publisher Full Text\n\nWolfsen HC, Crook JE, Krishna M, et al.: Prospective, controlled tandem endoscopy study of narrow band imaging for dysplasia detection in Barrett's Esophagus. Gastroenterology. 2008; 135(1): 24–31. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nSharma P, Bansal A, Mathur S, et al.: The utility of a novel narrow band imaging endoscopy system in patients with Barrett's esophagus. Gastrointest Endosc. 2006; 64(2): 167–75. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nSingh M, Bansal A, Curvers WL, et al.: Observer agreement in the assessment of narrowband imaging system surface patterns in Barrett's esophagus: a multicenter study. Endoscopy. 2011; 43(9): 745–51. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nSong J, Zhang J, Wang J, et al.: Meta-analysis of the effects of endoscopy with narrow band imaging in detecting dysplasia in Barrett's esophagus. Dis Esophagus. 2015; 28(6): 560–6. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nLongcroft-Wheaton G, Bhandari P: A review of image-enhanced endoscopy in the evaluation of colonic polyps. Expert Rev Gastroenterol Hepatol. 2014; 8(3): 267–81. PubMed Abstract \| Publisher Full Text\n\nHoffman A, Korczynski O, Tresch A, et al.: Acetic acid compared with i-scan imaging for detecting Barrett's esophagus: a randomized, comparative trial. Gastrointest Endosc. 2014; 79(1): 46–54. PubMed Abstract \| Publisher Full Text\n\nVerna C, Feyles E, Lorenzi L, et al.: I-SCAN targeted versus random biopsies in Barrett's oesophagus. Dig Liver Dis. 2014; 46(2): 131–4. PubMed Abstract \| Publisher Full Text\n\nCamus M, Coriat R, Leblanc S, et al.: Helpfulness of the combination of acetic acid and FICE in the detection of Barrett's epithelium and Barrett's associated neoplasias. World J Gastroenterol. 2012; 18(16): 1921–5. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nKara MA, Peters FP, Ten Kate FJ, et al.: Endoscopic video autofluorescence imaging may improve the detection of early neoplasia in patients with Barrett's esophagus. Gastrointest Endosc. 2005; 61(6): 679–85. PubMed Abstract \| Publisher Full Text\n\nBorovicka J, Fischer J, Neuweiler J, et al.: Autofluorescence endoscopy in surveillance of Barrett's esophagus: a multicenter randomized trial on diagnostic efficacy. Endoscopy. 2006; 38(9): 867–72. PubMed Abstract \| Publisher Full Text\n\nCurvers WL, Alvarez Herrero L, Wallace MB, et al.: Endoscopic tri-modal imaging is more effective than standard endoscopy in identifying early-stage neoplasia in Barrett's esophagus. Gastroenterology. 2010; 139(4): 1106–14. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nCurvers WL, van Vilsteren FG, Baak LC, et al.: Endoscopic trimodal imaging versus standard video endoscopy for detection of early Barrett's neoplasia: a multicenter, randomized, crossover study in general practice. Gastrointest Endosc. 2011; 73(2): 195–203. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nCanto MI, Setrakian S, Willis J, et al.: Methylene blue-directed biopsies improve detection of intestinal metaplasia and dysplasia in Barrett's esophagus. Gastrointest Endosc. 2000; 51(5): 560–8. PubMed Abstract \| Publisher Full Text\n\nWo JM, Ray MB, Mayfield-Stokes S, et al.: Comparison of methylene blue-directed biopsies and conventional biopsies in the detection of intestinal metaplasia and dysplasia in Barrett's esophagus: a preliminary study. Gastrointest Endosc. 2001; 54(3): 294–301. PubMed Abstract \| Publisher Full Text\n\nRagunath K, Krasner N, Raman VS, et al.: A randomized, prospective cross-over trial comparing methylene blue-directed biopsy and conventional random biopsy for detecting intestinal metaplasia and dysplasia in Barrett's esophagus. Endoscopy. 2003; 35(12): 998–1003. PubMed Abstract \| Publisher Full Text\n\nNgamruengphong S, Sharma VK, Das A: Diagnostic yield of methylene blue chromoendoscopy for detecting specialized intestinal metaplasia and dysplasia in Barrett's esophagus: a meta-analysis. Gastrointest Endosc. 2009; 69(6): 1021–8. PubMed Abstract \| Publisher Full Text\n\nLongcroft-Wheaton G, Duku M, Mead R, et al.: Acetic acid spray is an effective tool for the endoscopic detection of neoplasia in patients with Barrett's esophagus. Clin Gastroenterol Hepatol. 2010; 8(10): 843–7. PubMed Abstract \| Publisher Full Text\n\nPohl J, Pech O, May A, et al.: Incidence of macroscopically occult neoplasias in Barrett's esophagus: are random biopsies dispensable in the era of advanced endoscopic imaging? Am J Gastroenterol. 2010; 105(11): 2350–6. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nBhandari P, Kandaswamy P, Cowlishaw D, et al.: Acetic acid-enhanced chromoendoscopy is more cost-effective than protocol-guided biopsies in a high-risk Barrett's population. Dis Esophagus. 2012; 25(5): 386–92. PubMed Abstract \| Publisher Full Text\n\nTholoor S, Bhattacharyya R, Tsagkournis O, et al.: Acetic acid chromoendoscopy in Barrett's esophagus surveillance is superior to the standardized random biopsy protocol: results from a large cohort study (with video). Gastrointest Endosc. 2014; 80(3): 417–24. PubMed Abstract \| Publisher Full Text\n\nClinicalTrials.gov Identifier: NCT02407392. Reference Source\n\nPech O, May A, Manner H, et al.: Long-term efficacy and safety of endoscopic resection for patients with mucosal adenocarcinoma of the esophagus. Gastroenterology. 2014; 146(3): 652–660.e1. PubMed Abstract \| Publisher Full Text\n\nCobb MJ, Hwang JH, Upton MP, et al.: Imaging of subsquamous Barrett's epithelium with ultrahigh-resolution optical coherence tomography: a histologic correlation study. Gastrointest Endosc. 2010; 71(2): 223–30. PubMed Abstract \| Publisher Full Text\n\nKiesslich R, Gossner L, Goetz M, et al.: In vivo histology of Barrett's esophagus and associated neoplasia by confocal laser endomicroscopy. Clin Gastroenterol Hepatol. 2006; 4(8): 979–87. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nIsenberg G, Sivak MV Jr, Chak A, et al.: Accuracy of endoscopic optical coherence tomography in the detection of dysplasia in Barrett's esophagus: a prospective, double-blinded study. Gastrointest Endosc. 2005; 62(6): 825–31. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nGupta A, Attar BM, Koduru P, et al.: Utility of confocal laser endomicroscopy in identifying high-grade dysplasia and adenocarcinoma in Barrett's esophagus: a systematic review and meta-analysis. Eur J Gastroenterol Hepatol. 2014; 26(4): 369–77. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nEnzinger PC, Mayer RJ: Esophageal cancer. N Engl J Med. 2003; 349(23): 2241–52. PubMed Abstract \| Publisher Full Text\n\nStein HJ, Feith M, Bruecher BL, et al.: Early esophageal cancer: pattern of lymphatic spread and prognostic factors for long-term survival after surgical resection. Ann Surg. 2005; 242(4): 566–73; discussion 573–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThomas P, Doddoli C, Neville P, et al.: Esophageal cancer resection in the elderly. Eur J Cardiothorac Surg. 1996; 10(11): 941–6. PubMed Abstract \| Publisher Full Text\n\nBirkmeyer JD, Stukel TA, Siewers AE, et al.: Surgeon volume and operative mortality in the United States. N Engl J Med. 2003; 349(22): 2117–27. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nTanabe S, Koizumi W, Kokutou M, et al.: Usefulness of endoscopic aspiration mucosectomy as compared with strip biopsy for the treatment of gastric mucosal cancer. Gastrointest Endosc. 1999; 50(6): 819–22. PubMed Abstract \| Publisher Full Text\n\nEll C, May A, Gossner L, et al.: Endoscopic mucosal resection of early cancer and high-grade dysplasia in Barrett's esophagus. Gastroenterology. 2000; 118(4): 670–7. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nManner H, Rabenstein T, Pech O, et al.: Ablation of residual Barrett's epithelium after endoscopic resection: a randomized long-term follow-up study of argon plasma coagulation vs. surveillance (APE study). Endoscopy. 2014; 46(1): 6–12. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nOrman ES, Li N, Shaheen NJ: Efficacy and durability of radiofrequency ablation for Barrett's Esophagus: systematic review and meta-analysis. Clin Gastroenterol Hepatol. 2013; 11(10): 1245–55. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nHaidry RJ, Butt MA, Dunn JM, et al.: Improvement over time in outcomes for patients undergoing endoscopic therapy for Barrett's oesophagus-related neoplasia: 6-year experience from the first 500 patients treated in the UK patient registry. Gut. 2015; 64(8): 1192–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChedgy F, Basford PJ, Bhattacharyya R, et al.: Tu1569 Acetic acid guided endoscopic resection of dysplastic Barrett’s epithelium in a large UK series - the cheaper alternative to RFA? Gastrointest Endosc. 2015; 81(5): AB513. Publisher Full Text\n\nGhorbani S, Tsai FC, Greenwald BD, et al.: Safety and efficacy of endoscopic spray cryotherapy for Barrett's dysplasia: results of the National Cryospray Registry. Dis Esophagus. 2015. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nNeuhaus H, Terheggen G, Rutz EM, et al.: Endoscopic submucosal dissection plus radiofrequency ablation of neoplastic Barrett's esophagus. Endoscopy. 2012; 44(12): 1105–13. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nChevaux JB, Piessevaux H, Jouret-Mourin A, et al.: Clinical outcome in patients treated with endoscopic submucosal dissection for superficial Barrett's neoplasia. Endoscopy. 2015; 47(2): 103–12. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nProbst A, Aust D, Märkl B, et al.: Early esophageal cancer in Europe: endoscopic treatment by endoscopic submucosal dissection. Endoscopy. 2015; 47(2): 113–21. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nChedgy F, Bhattacharyya R, Kandiah K, et al.: Prospective comparison of EMR vs ESD in Barrett’s neoplasia: are we too afraid of knives in the oesophagus. UEGW. 2015. Reference Source\n\nTerheggen G, Rutz E, Vieth M, et al.: A prospective randomized controlled trial on endoscopic mucosal resection (EMR) versus water-jet assisted endoscopic submucosal dissection (WESD) for early Barrett’s neoplasia. DDW. 2015. Reference Source\n\nGiacchino M, Bansal A, Kim RE, et al.: Clinical utility and interobserver agreement of autofluorescence imaging and magnification narrow-band imaging for the evaluation of Barrett's esophagus: a prospective tandem study. Gastrointest Endosc. 2013; 77(5): 711–8. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nEvans JA, Poneros JM, Bouma BE, et al.: Optical coherence tomography to identify intramucosal carcinoma and high-grade dysplasia in Barrett's esophagus. Clin Gastroenterol Hepatol. 2006; 4(1): 38–43. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation" }	[ { "id": "12149", "date": "28 Jan 2016", "name": "Oliver Pech", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12150", "date": "28 Jan 2016", "name": "Ian Beales", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-113
https://f1000research.com/articles/5-112/v1	28 Jan 16	{ "type": "Review", "title": "Improved Cardiovascular Disease Outcomes in Older Adults", "authors": [ "Daniel E. Forman", "Karen Alexander", "Ralph G. Brindis", "Anne B. Curtis", "Mathew Maurer", "Michael W. Rich", "Laurence Sperling", "Nanette K. Wenger", "Karen Alexander", "Ralph G. Brindis", "Anne B. Curtis", "Mathew Maurer", "Michael W. Rich", "Laurence Sperling", "Nanette K. Wenger" ], "abstract": "Longevity is increasing and the population of older adults is growing. The biology of aging is conducive to cardiovascular disease (CVD), such that prevalence of coronary artery disease, heart failure, valvular heart disease, arrhythmia and other disorders are increasing as more adults survive into old age. Furthermore, CVD in older adults is distinctive, with management issues predictably complicated by multimorbidity, polypharmacy, frailty and other complexities of care that increase management risks (e.g., bleeding, falls, and rehospitalization) and uncertainty of outcomes. In this review, state-of-the-art advances in heart failure, acute coronary syndromes, transcatheter aortic valve replacement, atrial fibrillation, amyloidosis, and CVD prevention are discussed. Conceptual benefits of treatments are considered in relation to the challenges and ambiguities inherent in their application to older patients.", "keywords": [ "Geriatric", "Cardiovascular", "HFpEF" ], "content": "Introduction\n\nThe unprecedented growth of the elderly population throughout the world is evolving as a challenge for clinicians, governments, and health policy guidance. Older adults differ from those typically studied in clinical trials, efficacy and outcomes of care often remain ambiguous for older patients, and growing healthcare cost burdens for older adults are a related concern. In this review, we highlight some key advances associated with improved outcomes that have recently occurred in the management of older adults with cardiovascular disease.\n\n\nDemographics\n\nThe population of older adults is growing rapidly, particularly as longevity is also increasing. In the United States, the population aged 65 and over is projected to be 83.7 million by 2050, almost double the 43.1 million in 20121. The numbers of those who are very old (≥85 years) is growing the most rapidly. On a global level, the population ≥85 years is projected to increase 151% between 2005 and 2030, compared to the increase of only 104% in the population aged ≥65 years and only 21% for the population under 652. Similar growth of elderly populations is occurring in both developed and developing countries. Between 2012 and 2030, the percentages of adults older than 65 years are expected to increase 33.2% in Japan, 27.9% in Germany, and 25.5% in Italy3. In China, the world’s most populous country of 1.4 billion, the subgroup aged ≥65 years is expected to increase by 17.2% by 2030 and 26.8% by 2050, or stated as numbers, 350 million Chinese (a magnitude that is greater than the total US population) will soon be senior adults3.\n\n\nDistinctive implications of aging\n\nGeriatric features often transform healthcare challenges and are important concerns for clinical decision-making. Whereas cardiovascular management standards are fundamentally oriented to specific cardiac diseases, older adults typically present with multiple disease states occurring concurrently such that presentation and management of the cardiac issues are inherently more complex4. In some cases, multimorbid disease states lead to compounding instabilities (e.g. heart failure [HF] and renal failure). In other cases, multimorbidities give rise to new disease states (e.g. HF and preserved ejection fraction [HFpEF] arising from the substrate of metabolic disease and inflammation)5. Non-cardiac comorbid conditions also underlie much of the morbidity and rehospitalizations associated with HF, particularly HFpEF6. Geriatric syndromes are part of multimorbidity; frailty, sarcopenia, cognitive decline, and other age-related dimensions of health fundamentally compound multimorbidity management complexities of cardiac conditions7. These insights highlight the importance of a holistic approach in relation to HF as well as other cardiovascular diseases (CVDs) and call attention to the wide range of idiosyncratic capacities, vulnerabilities, and therapeutic objectives that typically distinguish one older cardiac patient from another. Similarly, typical cardiac medications (e.g. angiotensin-converting enzyme [ACE] inhibitors) must be regarded not only in terms of their evidence-based cardiac benefits but also in respect to the iatrogenesis they more easily (even predictably) provoke among older adults (e.g. syncope and falls) in the context of hypotension, balance or vision impairment, sleep deprivation, alcohol use, and other common conditions in an older patient population8.\n\n\nHeart failure\n\nTherapeutic options for the management of HF in older adults include medications, devices, and behavioral interventions. The PARADIGM-HF trial randomized 8442 patients with symptomatic HF and a left ventricular ejection fraction ≤40% to the ACE inhibitor enalapril or to the combination of the angiotensin-receptor blocker valsartan and the neprilysin inhibitor sacubitril9. Compared to enalapril, valsartan/sacubitril was associated with significant reductions in cardiovascular death or hospitalization for HF, as well as all-cause mortality, cardiovascular mortality, HF hospitalizations, and HF-related symptoms. Patients ≥65 years and those ≥75 years derived similar benefits from combination therapy, as did younger patients. Based on these findings, it is anticipated that valsartan/sacubitril will be rapidly incorporated into the management of older and younger patients with HF and reduced ejection fraction (HFrEF).\n\nIn contrast to HFrEF, the treatment of patients with HFpEF, the vast majority of whom are elderly, remains problematic. In the Treatment of Preserved Cardiac Function Heart Failure With an Aldosterone Antagonist (TOPCAT) trial, spironolactone failed to reduce the primary outcome of cardiovascular death, aborted cardiac arrest, or HF hospitalizations10. Spironolactone reduced HF hospitalizations at the expense of increased rates of hyperkalemia and elevated serum creatinine. Hence, the role of spironolactone in the management of HFpEF remains uncertain.\n\nIn the realm of devices, the value of implantable cardioverter-defibrillators (ICDs) in patients ≥75 years of age remains controversial, and selection of older patients for ICDs must be individualized11. Mechanical circulatory support (MCS) appears to be associated with improved outcomes in carefully selected older adults with advanced HF, albeit with increased risk of complications compared to younger patients12. Pre-morbid frailty is a strong predictor of worse outcomes in older adults receiving MCS13.\n\nIn the domain of behavioral interventions, there is a need for novel approaches that address HF not in isolation, but rather in the context of a complex array of comorbid conditions and heterogeneous personal preferences regarding goals of care in order to optimize patient-centered outcomes14. In addition, the management of HF in long-term care facilities and at the end-of-life now raises special considerations to maximize quality of life and reduce suffering15,16.\n\n\nAcute coronary syndrome\n\nRecent developments in antithrombotic therapy as well as better information on frailty and outcomes have benefitted older adults with acute coronary syndrome (ACS). A clinical challenge in older adults presenting with a myocardial infarction (MI) or undergoing percutaneous coronary intervention (PCI) is the indication for multiple antithrombotic agents (e.g. atrial fibrillation [AF] or valvular disease). Triple therapy increases the rate of major bleeding, an outcome already common among older adults. Two recent European registry studies further the understanding of antithrombotic therapy in patients with AF with MI and/or undergoing PCI. Among older adults requiring oral anticoagulation presenting with MI or undergoing PCI, clopidogrel added to oral anticoagulant therapy was as effective as triple therapy (including aspirin) in preventing MI/coronary death, ischemic stroke, and bleeding17. In another study, among patients requiring oral anticoagulation undergoing PCI (80% ACS, 65% drug-eluting stent and 30% bare metal stent), use of clopidogrel without aspirin was also associated with less bleeding and no increase in thrombotic events during follow up18. A supporting secondary analysis from a large AF clinical trial found the oral anticoagulant apixaban had similar beneficial effects on stroke, MI, and major bleeding, with or without aspirin, compared with warfarin19. Ongoing research is exploring the ability to omit aspirin in the setting of an effective oral anticoagulant. These new and safer antithrombotic strategies will be an important advance for high-risk individuals with coronary artery disease and indications for oral anticoagulation.\n\nOlder adults are at high risk of adverse outcomes following MI, but a Medicare linkage analysis from the CRUSADE Registry provides evidence that most mortality occurs early and not in the context of rehospitalization. Contrary to expectations, rehospitalization rate did not rise substantially with age, in part due to competing mortality. Rehospitalizations which did occur were often for non-cardiac diagnoses, underscoring the multimorbidity also present in the older population with a coronary event20. The TaRgeted platelet Inhibition to cLarify the Optimal strateGy to medicallY manage Acute Coronary Syndromes (TRILOGY ACS) trial, which randomized 9326 patients with MI planned for medical management to prasugrel or clopidogrel, added frailty assessment at baseline for patients aged ≥65 years21. The modified Fried score classified 23.0% of the older adults in this trial as pre-frail (1–2 items) and 4.7% as frail (≥3 items). After adjustment, frailty remained independently associated with the composite of cardiovascular death, MI, or stroke: pre-frail vs. not-frail, hazard ratio (HR): 1.33; 95% confidence interval (CI): 1.15–1.54; p<0.001; frail vs. not-frail, HR: 1.52; 95% CI: 1.18–1.98; p=0.002. Frailty among registry populations is higher than in clinical trials, so care delivery models should add early targeted follow up in older adults after MI, particularly for those with multimorbidity or frailty. Future studies will benefit from collecting frailty data to allow comparative effectiveness and outcomes comparisons across older MI or PCI populations.\n\n\nTranscatheter aortic valve replacement\n\nTranscatheter aortic valve replacement (TAVR) represents an innovative interventional technology that provides a non-surgical alternative for the management of severe aortic stenosis (AS) that has particular relevance for the elderly and the very elderly populations. The prevalence of AS in the US is estimated to be greater than 4–5% of those over 75 years of age. Approximately 50% of all US patients and over 75% of those over 80 years of age having clinical indications for surgical aortic valve replacement (AVR) are not being surgically treated due to either the lack of referral by clinicians or patient/family refusal. The use of TAVR has been projected to make substantial inroads in this underserved population, particularly those who are very elderly or deemed at high surgical risk.\n\nThe key randomized clinical trials (RCTs) PARTNER A & B (utilizing Edwards’ Sapien balloon expandable AVR) along with the CoreValve (Medtronic’s self-expanding percutaneous AVR) study of high-risk patients conclusively demonstrated improved long-term patient survival employing TAVR compared to sAVR (standard surgical AVR) with acceptable stroke and bleeding rates for patients felt either surgically inoperable or at very high surgical risk. The median age of TAVR implantation in these study cohorts was approximately 84 years22,23.\n\nTAVR had been available for clinical use in over 31 countries prior to United States FDA approval in 2011. To help ensure a rational diffusion of TAVR’s innovative technology partnerships between the Food and Drug Administration (FDA), the Center for Medicare and Medical Services (CMS) along with cardiovascular professional societies led by the American College of Cardiology (ACC) and the Society for Thoracic Surgery (STS) collaborated in the creation of critical Post Approval Studies, CMS Coverage Determination Criteria, a multi-societal competency document for institutional and professional utilization of TAVR technology accompanied by an expert consensus document and clinical practice guidelines for TAVR clinical use. STS and ACC have created the STS/ACC transcatheter valve therapy (TVT) Registry where all commercial TAVR implants are required to be enrolled by hospitals to satisfy CMS coverage and payment requirements24–27. As of spring 2015, over 30,000 TAVR patients from approximately 350 institutions have been entered into the TVT Registry.\n\nThe TVT Registry data has been instrumental in assuring quality of care along with providing the infrastructure for performing Post Approval Studies, Investigational Device Exemption Studies, and Post Market Surveillance28,29. The Registry allowed earlier FDA approval than would be possible through previous RCT mechanisms for “Valve-in-Valve” TAVR use and also for alternative access use in high-risk and surgically inoperable patients. The TVT Registry demonstrated 7.0% 30-day mortality, and 23.7% 1-year mortality with a stroke rate of 4.1%30.\n\nOf great importance for assessing clinical decision-making for elderly patients with AS, the TVT Registry is collecting both baseline and longitudinal data on frailty and activity limitation assessments through 5-meter walk tests and the Kansas City Cardiomyopathy Questionnaire (KCCQ). In addition, the TVT Registry is creating a risk-adjusted mortality model that will hopefully soon be transformed into a predictive outcome tool to help clinicians, patients, and families make informed decisions in the management of elderly and very elderly patients with AS.\n\n\nAtrial fibrillation\n\nThe median age of patients with AF is 75 years, with a prevalence of about 9% in the elderly31. Stroke stands out as one of the greatest risks attributable to this arrhythmia. The 2014 American Heart Association/ACC/Heart Rhythm Society (AHA/ACC/HRS) guideline32 recommends using the CHA2DS2-VASc (Congestive HF [CHF], Hypertension, Age ≥75 years, Diabetes, Stroke/Transient ischemic attack [TIA], Vascular disease, Age 65–74, Sex category [female]) score to assess stroke risk. The guideline recommends oral anticoagulant therapy for AF patients with a CHA2DS2-VASc >2 using warfarin or one of the new oral anticoagulants (NOACs) dabigatran, rivaroxaban, or apixaban. Edoxaban is another NOAC that was approved for use after the guideline was published, but it has efficacy that is comparable to those approved earlier.\n\nAs age >75 years leads to a minimum CHA2DS2-VASc score of 2, many emphasize the value of anticoagulation for most elderly patients with AF and decry the underuse of such vital therapy. Nonetheless, this still remains an issue of debate amidst age-related intricacies of care, i.e. intrinsic bleeding risks are often compounded by frailty, multimorbidity, polypharmacy, falls, and other management complexities.\n\nThere have been several studies to estimate the risk/benefit ratio of treating elderly AF patients with oral anticoagulation, including the Birmingham Atrial Fibrillation Treatment of the Aged (BAFTA) study33, which randomized individuals aged ≥75 years of age to aspirin or warfarin based on physician discretion. Stroke/systemic embolism (SE) occurred at a rate of 1.8%/year in warfarin-treated patients versus 3.8%/year in the aspirin group (HR 0.48; 95% CI, 0.28–0.80), with no difference in the rates of major hemorrhage (1.9 vs. 2.2%, respectively). The risk for bleeding with warfarin therapy in patients >80 years of age has been estimated to range from 1.63% to 13.1% per year34,35.\n\nThe NOACs have generated much excitement in the field of AF management, as they enable relatively simplified treatment compared to warfarin (e.g. reduced need for blood testing and no dietary restrictions). NOACs that include both direct thrombin inhibitors (dabigatran) and factor X inhibitors (rivaroxaban, apixaban, and edoxaban) are now available as alternatives to warfarin. All have been shown to be non-inferior to warfarin with respect to stroke and SE, with comparable rates of bleeding36–39. The mean age of patients in these studies was approximately 70 years, with about one-third of the patients being over 75 years.\n\nA recent meta-analysis of the efficacy and harm of the NOACs for prevention of stroke in AF and secondary prevention of thromboembolism found that the efficacy of each of the NOACs was equal or superior to warfarin in elderly patients40. Dabigatran was associated with a higher risk of gastrointestinal bleeding and a lower risk of intracranial bleeding. A significantly lower risk of major bleeding compared to warfarin was found for apixaban (odds ratio 0.63, 95% CI 0.51–0.77) and edoxaban 60 mg (0.81, 0.67–0.98) and 30 mg (0.46, 0.38–0.57), while rivaroxaban showed similar risks.\n\n\nAmyloidosis\n\nCardiac amyloid has traditionally been considered a rare cardiovascular condition. However, emerging data demonstrate that transthyretin cardiac amyloidosis (ATTR), formerly known as senile cardiac amyloidosis, caused by misfolded monomers or oligomers of the protein transthyretin (TTR), is a common cause of HFpEF. Indeed recent autopsy studies in HFpEF subjects (mean age 76 years) showed 21% had amyloid deposits and the prevalence was greater in those ≥75 years (32%) vs. <75 years (8%). Furthermore, only 20% of the HFpEF patients with amyloid at autopsy had received a pre-morbid diagnosis41.\n\nBone isotopes, commonly used in bone scans, have a high sensitivity and specificity (>90%) for identifying TTR cardiac amyloid due to either wild-type TTR or a mutant allele42,43. While over 20 mutations can cause TTR cardiac amyloidosis, the Val122Ile mutation (substitution of isoleucine for valine at position 122) is the most common in the United States with a particularly high frequency (prevalence of 3.4% to 3.9%) in African-Americans44. The Thr60Ala mutation (substitution of alanine for threonine at position 60) is also notably common in individuals of Irish descent and is referred to as the Appalachian mutation. These genetic conditions have an age-dependent penetrance, with males over the age of 60 years being most commonly affected.\n\nHistorically, cardiac amyloid was difficult to diagnose because it can masquerade as other cardiovascular disorders45 and usually required endomyocardial biopsy. The diagnosis has become easier with the discovery that bone isotopes have a very high sensitivity and specificity for distinguishing ATTR cardiac amyloid (both mutant and wild-type) from light chain (AL) cardiac amyloid46 and other types of cardiomyopathy that mimic amyloid (e.g. hypertrophic cardiomyopathy). Differentiating TTR from AL cardiac amyloid has important prognostic, management, counseling, and therapeutic implications. These nuclear medicine techniques have the potential to dramatically alter the outcomes of patients with ATTR cardiac amyloidosis, particularly because new therapeutic options are looming.\n\nEmerging therapeutic strategies to treat ATTR amyloid are focused on small molecules to stabilize the transthyretin tetramer and agents to silence TTR production. Phase II clinical trial data47 suggest that tafamidis was generally well tolerated and stabilized TTR, leading to a phase III clinical trial that has completed enrollment. TTR silencers using either small interfering RNA (siRNA) or oligonucleotides specific for silencing TTR production have shown remarkable ability to lower TTR to >80% of normal levels in subjects with TTR cardiac amyloidosis. Collectively, such data remind us that TTR cardiac amyloid is certainly not rare and hopefully not unmodifiable.\n\n\nPreventive care\n\nRecent guidelines for cardiovascular prevention importantly emphasize that older adults are at highest risk for atherosclerotic cardiovascular events. Paradoxically, however, strategies focused on cardiovascular prevention are often underutilized in this subpopulation48. As the elderly also have the greatest burden of comorbidities, polypharmacy, and potential for medication-related side effects, the need for comprehensive and collaborative clinician-patient management is imperative49.\n\nPotential overtreatment of hypertension, in addition to inadequate control, may cause adverse outcomes in the elderly, who are more likely to have target organ damage. Considerable debate has arisen from the guideline recommendations made by the panel members appointed to JNC 8 (Eighth Joint National Committee) that blood pressure should be reduced to less than 150/90 mm Hg in adults aged 60 years and older without diabetes or chronic kidney disease50. In support of the ACC/AHA 2011 Expert Consensus document on hypertension in the elderly51 that recommended blood pressure be reduced to less than 140/90 in adults aged 60–79 (and a systolic pressure of 140–145 if tolerated in adults aged 80 and older) are data from REGARDS (REasons for Geographic and Racial Differences in Stroke), which reported that optimal blood pressure for elderly patients on antihypertensive therapy for the reduction of cardiovascular events and all-cause mortality was less than 140/90 mm Hg52.\n\nSeveral recommended caveats for the care of older adults are highlighted by the ACC/AHA 2013 guidelines for the treatment of blood cholesterol, including the use of moderate-intensity statin therapy in adults >75 years of age with established atherosclerotic CVD (ASCVD). The ASCVD pooled cohort risk estimator does not provide for risk assessment in patients >79 years of age53. When initiating statin therapy, the risk of transitioning to diabetes should be discussed, although this risk is small in relation to the potential benefits of statin medications. Importantly, there is no definitive evidence that statins lead to cognitive decline. The available data on the use of proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor therapy in those of advanced age is promising, as low-density lipoprotein (LDL)-C is potently reduced with a side effect profile similar to that of placebo54.\n\nLifestyle approaches remain the cornerstone for cardiovascular prevention in the elderly. Among persons aged 55 to 80 (mean age 67) at increased cardiovascular risk, a Mediterranean diet supplemented with extra-virgin olive oil or mixed nuts reduced the incidence of major cardiovascular events by 30%55. Emphasizing a goal of living longer and better, a Mediterranean lifestyle including dietary pattern, moderate alcohol intake, regular physical activity, and not smoking was associated with a 60% reduction in all-cause mortality in elderly Europeans aged 70–9056.\n\n\nThe value of geriatric measures in patient-centered decision-making for clinical cardiovascular problems\n\nIn general, medications, procedures, and other components of cardiovascular care are more likely to entail trade-offs at elderly age. Polypharmacy, pain, fatigue, confusion, dysgeusia, excessive time for recovery, and other sequelae may detract from (and sometimes even supersede) intended therapeutic benefits57,58. Whereas life prolongation is the overriding goal of therapy in the young, for many seniors issues of independence, quality of life, and functional capacity are often more important, and this may determine therapeutic choices that contrast with those of younger populations. This broader spectrum of clinical objectives and the high susceptibility to iatrogenesis are driving rationales for a growing emphasis on principles of shared decision-making between older patients and their clinicians57,58.\n\nOther age-related issues that commonly affect caregiving of older cardiac patients include frailty, altered body composition (diminished lean body mass and increased interstitial fat), and changes in cognition. Frailty is often assessed as a constellation of phenotypic changes that relate to a state of increased vulnerability, i.e. slowing, weakening, weight loss, exhaustion, and reduced activity59. Patients with three or more of these measures are considered frail and are generally at increased risk for disease but also for poor tolerance of therapy. Frailty therefore fundamentally factors into therapeutic decisions and management strategies. Sarcopenia or reduced lean body mass is a subpart of frailty and directly detracts from physical capacity and vital resiliency to recover60. Diminished cognition, particularly executive function, is similarly insidious and detrimental, and complicating with regard to decision-making and adherence, and even in respect to the quality of life benefits that are achievable by therapy61.", "appendix": "Competing interests\n\n\n\nDr. Forman: No COI\n\nRalph Brindis: No COI\n\nAnne Curtis:\n\n• Advisory board:\n\nDaiichi Sankyo\n\nPfizer, Inc.\n\nJanssen Pharmaceuticals\n\nSt. Jude Medical\n\n• Honoraria:\n\nSt. Jude Medical\n\nMedtronic, Inc.\n\n• Consultant:\n\nMedtronic, Inc.\n\nMichael Rich: No COI\n\nLaurence Sperling: No COI\n\nNanette Wenger: No COI\n\n\nGrant information\n\nMathew Maurer’s institution, Columbia University Medical Center, receives funding for research and serving on advisory boards and DSMBs from Pfizer Inc., Alnylam Pharmaceuticals Inc., ISIS Pharmaceuticals and Prothena Inc.\n\n\nReferences\n\nOrtman JM, Velkoff VA, Hogan H: An Aging Nation: The older population in the United States. 2014. Reference Source\n\nNational Institute of Aging. 2015. Reference Source\n\nU.S. Census Bureau. International Data Base. 2009. Reference Source\n\nBell SP, Orr NM, Dodson JA, et al.: What to Expect From the Evolving Field of Geriatric Cardiology. J Am Coll Cardiol. 2015; 66(11): 1286–99. PubMed Abstract \| Publisher Full Text\n\nPaulus WJ, Tschöpe C: A novel paradigm for heart failure with preserved ejection fraction: comorbidities drive myocardial dysfunction and remodeling through coronary microvascular endothelial inflammation. J Am Coll Cardiol. 2013; 62(4): 263–71. PubMed Abstract \| Publisher Full Text\n\nMentz RJ, Kelly JP, von Lueder TG, et al.: Noncardiac comorbidities in heart failure with reduced versus preserved ejection fraction. J Am Coll Cardiol. 2014; 64(21): 2281–93. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChaudhry SI, Wang Y, Gill TM, et al.: Geriatric conditions and subsequent mortality in older patients with heart failure. J Am Coll Cardiol. 2010; 55(4): 309–16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChang VC, Do MT: Risk factors for falls among seniors: implications of gender. Am J Epidemiol. 2015; 181(7): 521–31. PubMed Abstract \| Publisher Full Text\n\nMcMurray JJ, Packer M, Desai AS, et al.: Angiotensin-neprilysin inhibition versus enalapril in heart failure. N Engl J Med. 2014; 371(11): 993–1004. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nPitt B, Pfeffer MA, Assmann SF, et al.: Spironolactone for heart failure with preserved ejection fraction. N Engl J Med. 2014; 370(15): 1383–92. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nBarra S, Providência R, Paiva L, et al.: Implantable cardioverter-defibrillators in the elderly: rationale and specific age-related considerations. Europace. 2015; 17(2): 174–86. PubMed Abstract \| Publisher Full Text\n\nRosenbaum AN, John R, Liao KK, et al.: Survival in elderly patients supported with continuous flow left ventricular assist device as bridge to transplantation or destination therapy. J Card Fail. 2014; 20(3): 161–7. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nFlint KM, Matlock DD, Lindenfeld J, et al.: Frailty and the selection of patients for destination therapy left ventricular assist device. Circ Heart Fail. 2012; 5(2): 286–93. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMurad K, Kitzman DW: Frailty and he selection of patients for destination therapy for left ventricular assist device. Circ Hrt Fail. 2012; 5: 246–93.\n\nJurgens CY, Goodlin S, Dolansky M, et al.: Heart failure management in skilled nursing facilities: a scientific statement from the American Heart Association and the Heart Failure Society of America. J Card Fail. 2015; 21(4): 263–99. PubMed Abstract \| Publisher Full Text\n\nWhellan DJ, Goodlin SJ, Dickinson MG, et al.: End-of-life care in patients with heart failure. J Card Fail. 2014; 20(2): 121–34. PubMed Abstract \| Publisher Full Text\n\nLamberts M, Gislason GH, Olesen JB, et al.: Oral anticoagulation and antiplatelets in atrial fibrillation patients after myocardial infarction and coronary intervention. J Am Coll Cardiol. 2013; 62(11): 981–9. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nDewilde WJ, Oirbans T, Verheugt FW, et al.: Use of clopidogrel with or without aspirin in patients taking oral anticoagulant therapy and undergoing percutaneous coronary intervention: an open-label, randomised, controlled trial. Lancet. 2013; 381(9872): 1107–15. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nAlexander JH, Lopes RD, Thomas L, et al.: Apixaban vs. warfarin with concomitant aspirin in patients with atrial fibrillation: insights from the ARISTOTLE trial. Eur Heart J. 2014; 35(4): 224–32. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nLopes RD, Gharacholou SM, Holmes DN, et al.: Cumulative incidence of death and rehospitalization among the elderly in the first year after NSTEMI. Am J Med. 2015; 128(6): 582–90. PubMed Abstract \| Publisher Full Text\n\nWhite HD, Westerhout CM, Alexander KP, et al.: Frailty is associated with worse outcomes in non-ST-segment elevation acute coronary syndromes: Insights from the TaRgeted platelet Inhibition to cLarify the Optimal strateGy to medicallY manage Acute Coronary Syndromes (TRILOGY ACS) trial. EEur Heart J Acute Cardiovasc Care. 2015; pii: 2048872615581502. PubMed Abstract \| Publisher Full Text\n\nSmith CR, Leon MB, Mack MJ, et al.: Transcatheter versus surgical aortic-valve replacement in high-risk patients. N Engl J Med. 2011; 364(23): 2187–98. 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J Am Coll Cardiol. 2012; 59(13): 1200–54. PubMed Abstract \| Publisher Full Text\n\nCarroll JD, Edwards FH, Marinac-Dabic D, et al.: The STS-ACC transcatheter valve therapy national registry: a new partnership and infrastructure for the introduction and surveillance of medical devices and therapies. J Am Coll Cardiol. 2013; 62(11): 1026–34. PubMed Abstract \| Publisher Full Text\n\nMack MJ, Brennan JM, Brindis R, et al.: Outcomes following transcatheter aortic valve replacement in the United States. JAMA. 2013; 310(19): 2069–77. PubMed Abstract \| Publisher Full Text\n\nHolmes DR Jr, Brennan JM, Rumsfeld JS, et al.: Clinical outcomes at 1 year following transcatheter aortic valve replacement. JAMA. 2015; 313(10): 1019–28. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nRosamond W, Flegal K, Furie K, et al.: Heart disease and stroke statistics--2008 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Circulation. 2008; 117(4): e25–146. PubMed Abstract \| Publisher Full Text\n\nJanuary CT, Wann LS, Alpert JS, et al.: 2014 AHA/ACC/HRS guideline for the management of patients with atrial fibrillation: executive summary: a report of the American College of Cardiology/American Heart Association Task Force on practice guidelines and the Heart Rhythm Society. Circulation. 2014; 130(23): 2071–104. PubMed Abstract \| Publisher Full Text\n\nMant J, Hobbs FD, Fletcher K, et al.: Warfarin versus aspirin for stroke prevention in an elderly community population with atrial fibrillation (the Birmingham Atrial Fibrillation Treatment of the Aged Study, BAFTA): a randomised controlled trial. Lancet. 2007; 370(9586): 493–503. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nPoli D, Antonucci E, Testa S, et al.: Bleeding risk in very old patients on vitamin K antagonist treatment: results of a prospective collaborative study on elderly patients followed by Italian Centres for Anticoagulation. Circulation. 2011; 124(7): 824–9. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nHylek EM, Evans-Molina C, Shea C, et al.: Major hemorrhage and tolerability of warfarin in the first year of therapy among elderly patients with atrial fibrillation. Circulation. 2007; 115(21): 2689–96. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nConnolly SJ, Ezekowitz MD, Yusuf S, et al.: Dabigatran versus warfarin in patients with atrial fibrillation. N Engl J Med. 2009; 361(12): 1139–51. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nPatel MR, Mahaffey KW, Garg J, et al.: Rivaroxaban versus warfarin in nonvalvular atrial fibrillation. N Engl J Med. 2011; 365(10): 883–91. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nGranger CB, Alexander JH, McMurray JJ, et al.: Apixaban versus warfarin in patients with atrial fibrillation. N Engl J Med. 2011; 365(11): 981–92. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nGiugliano RP, Ruff CT, Braunwald E, et al.: Edoxaban versus warfarin in patients with atrial fibrillation. N Engl J Med. 2013; 369(22): 2093–104. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nSharma M, Cornelius VR, Patel JP, et al.: Efficacy and Harms of Direct Oral Anticoagulants in the Elderly for Stroke Prevention in Atrial Fibrillation and Secondary Prevention of Venous Thromboembolism: Systematic Review and Meta-Analysis. Circulation. 2015; 132(3): 194–204. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nMohammed SF, Mirzoyev SA, Edwards WD, et al.: Left ventricular amyloid deposition in patients with heart failure and preserved ejection fraction. JACC Heart Fail. 2014; 2(2): 113–22. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGonzález-López E, Gallego-Delgado M, Guzzo-Merello G, et al.: Wild-type transthyretin amyloidosis as a cause of heart failure with preserved ejection fraction. Eur Heart J. 2015; 36(38): 2585–94. PubMed Abstract \| Publisher Full Text\n\nMaurer MS: Noninvasive Identification of ATTRwt Cardiac Amyloid: The Re-emergence of Nuclear Cardiology. Am J Med. 2015; 128(12): 1275–80. PubMed Abstract \| Publisher Full Text\n\nQuarta CC, Buxbaum JN, Shah AM, et al.: The amyloidogenic V122I transthyretin variant in elderly black Americans. N Engl J Med. 2015; 372(1): 21–9. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nRapezzi C, Lorenzini M, Longhi S, et al.: Cardiac amyloidosis: the great pretender. Heart Fail Rev. 2015; 20(2): 117–24. PubMed Abstract \| Publisher Full Text\n\nBokhari S, Castaño A, Pozniakoff T, et al.: 99mTc-pyrophosphate scintigraphy for differentiating light-chain cardiac amyloidosis from the transthyretin-related familial and senile cardiac amyloidoses. Circ Cardiovasc Imaging. 2013; 6(2): 195–201. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMaurer MS, Grogan DR, Judge DP, et al.: Tafamidis in transthyretin amyloid cardiomyopathy: effects on transthyretin stabilization and clinical outcomes. Circ Heart Fail. 2015; 8(3): 519–26. PubMed Abstract \| Publisher Full Text\n\nWilmot KA, Khan A, Krishnan S, et al.: Statins in the elderly: a patient-focused approach. Clin Cardiol. 2015; 38(1): 56–61. PubMed Abstract \| Publisher Full Text\n\nMartin SS, Sperling LS, Blaha MJ, et al.: Clinician-patient risk discussion for atherosclerotic cardiovascular disease prevention: importance to implementation of the 2013 ACC/AHA Guidelines. J Am Coll Cardiol. 2015; 65(13): 1361–8. PubMed Abstract \| Publisher Full Text\n\nJames PA, Oparil S, Carter BL, et al.: 2014 evidence-based guideline for the management of high blood pressure in adults: report from the panel members appointed to the Eighth Joint National Committee (JNC 8). JAMA. 2014; 311(5): 507–20. PubMed Abstract \| Publisher Full Text\n\nAronow WS, Fleg JL, Pepine CJ, et al.: ACCF/AHA 2011 expert consensus document on hypertension in the elderly: a report of the American College of Cardiology Foundation Task Force on Clinical Expert Consensus documents developed in collaboration with the American Academy of Neurology, American Geriatrics Society, American Society for Preventive Cardiology, American Society of Hypertension, American Society of Nephrology, Association of Black Cardiologists, and European Society of Hypertension. J Am Coll Cardiol. 2011; 57(20): 2037–114. PubMed Abstract \| Publisher Full Text\n\nBanach M, Bromfield S, Howard G, et al.: Association of systolic blood pressure levels with cardiovascular events and all-cause mortality among older adults taking antihypertensive medication. Int J Cardiol. 2014; 176(1): 219–26. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nStone NJ, Robinson JG, Lichtenstein AH, et al.: 2013 ACC/AHA guideline on the treatment of blood cholesterol to reduce atherosclerotic cardiovascular risk in adults: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines. Circulation. 2014; 129(25 Suppl 2): S1–45. PubMed Abstract \| Publisher Full Text\n\nKoren M, Rosenson R, Khan B, et al.: Ldl cholesterol reduction in elderly patients with the pcsk9 monoclonal antibody evolocumab (amg 145): a pooled analysis of 1779 patients in phase 2, 3 and open label extension studies. J Am Coll Cardiol. 2015; 65(10): A1366. Publisher Full Text\n\nEstruch R, Ros E, Salas-Salvadó J, et al.: Primary prevention of cardiovascular disease with a Mediterranean diet. N Engl J Med. 2013; 368(14): 1279–90. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nKnoops KT, Kim TB, de Groot LC, et al.: Mediterranean diet, lifestyle factors, and 10-year mortality in elderly European men and women: the HALE project. JAMA. 2004; 292(12): 1433–9. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nLorgunpai SJ, Grammas M, Lee DS, et al.: Potential therapeutic competition in community-living older adults in the U.S.: use of medications that may adversely affect a coexisting condition. PLoS One. 2014; 9(2): e89447. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCase SM, O'Leary J, Kim N, et al.: Older Adults' Recognition of Trade-Offs in Healthcare Decision-Making. J Am Geriatr Soc. 2015; 63(8): 1658–62. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nAfilalo J, Alexander KP, Mack MJ, et al.: Frailty assessment in the cardiovascular care of older adults. J Am Coll Cardiol. 2014; 63(8): 747–62. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMilte R, Crotty M: Musculoskeletal health, frailty and functional decline. Best Pract Res Clin Rheumatol. 2014; 28(3): 395–410. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nCannon JA, McMurray JJ, Quinn TJ, et al.: 'Hearts and minds': association, causation and implication of cognitive impairment in heart failure. Alzheimers Res Ther. 2015; 7(1): 22. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation" }	[ { "id": "12151", "date": "28 Jan 2016", "name": "Joseph Alpert", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12152", "date": "28 Jan 2016", "name": "Brian Olshansky", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12153", "date": "28 Jan 2016", "name": "Michael Gaziano", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-112
https://f1000research.com/articles/5-111/v1	27 Jan 16	{ "type": "Review", "title": "Structural Mechanisms of Hexameric Helicase Loading, Assembly, and Unwinding", "authors": [ "Michael A. Trakselis" ], "abstract": "Hexameric helicases control both the initiation and the elongation phase of DNA replication. The toroidal structure of these enzymes provides an inherent challenge in the opening and loading onto DNA at origins, as well as the conformational changes required to exclude one strand from the central channel and activate DNA unwinding. Recently, high-resolution structures have not only revealed the architecture of various hexameric helicases but also detailed the interactions of DNA within the central channel, as well as conformational changes that occur during loading. This structural information coupled with advanced biochemical reconstitutions and biophysical methods have transformed our understanding of the dynamics of both the helicase structure and the DNA interactions required for efficient unwinding at the replisome.", "keywords": [ "Hexameric Helicase", "DNA replication", "DNA unwinding", "Helicase Loading", "Helicase Assembly" ], "content": "Introduction and context\n\nCell proliferation relies on the exact replication of an organism’s genetic material in a rapid but precisely controlled and efficient manner. The process and mechanism of DNA replication directs targeted and repetitive enzymatic activities towards long linear polymers of DNA. Interestingly, organisms have evolved a number of toroidal DNA replication and repair protein complexes that can maintain repetitive catalysis by encircling the DNA substrate. These protein-DNA rotaxane-like systems have the intrinsic ability to be processive enzymes due to their topological linkage with the substrate. As such, they provide inherent challenges to the loading and encircling of DNA. The steps required for the loading and encircling of circular protein complexes onto DNA provides for a higher level of regulation, which is required to restrict cell cycle progression and control DNA replication initiation. Because of this, the most highly regulated component within the DNA replisome is the loading and activation of the hexameric helicase, which dictates both the initiation steps and the elongation rate of DNA replication. Even though the general toroidal hexameric helicase structure has been known for more than two decades, the mechanisms for loading, encircling, activating, and unwinding are only just being discovered. These recent advances have been primarily aided by higher resolution structures that include DNA, better mechanistic descriptions of the interactions of the helicase with each separated strand of single-stranded DNA (ssDNA), and higher order in vitro reconstitution of DNA replication systems. It is an exciting time to be a part of the hexameric helicase field as big questions regarding dynamic structure-function relationships with DNA are poised to be revealed.\n\n\nHexameric helicase architectural conservation\n\nAlthough the general architecture of hexameric DNA replication helicases is shared across organismal domains, there is strong evidence that classes of these enzymes have evolved independently for a role in DNA replication1. Although all hexameric helicases are members of the broader P-loop family of ATPases2, individual evolution of RecA domains gave rise to the superfamily (SF) 4 helicases including T4 gp41, T7 gp4, bacterial DnaB, and mitochondrial Twinkle3, while SF3, including SV40 Large T antigen (SV40 L-Tag) and papilloma virus E1, and SF6, including archaeal and eukaryotic minichromosome maintenance proteins (MCM), helicases came from an ATPases associated with a variety of cellular activities (AAA+) clade (Figure 1 & Table 1)4,5. Regardless of the origin, these systems have all converged on a common ring-shaped architecture wherein a central channel is used to repetitively engage and translocate along ssDNA during unwinding. Bacterial and phage SF4 helicases are perhaps the best studied and have contributed most to our understanding of DNA unwinding, but more recent emphases on SF3 and SF6 helicases are providing insight into structure-function relationships across SFs.\n\nHexameric helicases are shown from different domains (RecA or AAA+) and superfamilies (SFs) with associated unwinding polarity and references. View from the N-terminal domain (NTD) rotated 90° to visualize the lateral length from the C-terminal domain (CTD) to the NTD.\n\nHelicase activity requires the presence of both nucleotide triphosphates (NTPs)6 and Mg2+ for unwinding7–9. Although it is not known exactly how ATP hydrolysis directly drives DNA unwinding, it is likely to progress in a sequential manner, with each subunit driving conformational changes throughout the hexamer that contribute to unwinding polarity4,10–14. RecA-like helicases (SF4) translocate along ssDNA in the 5’ → 3’ direction, while AAA+ enzymes (SF3 and SF6) translocate in the 3’ → 5’ direction13,15–18. The core ATP binding and hydrolysis domains consist of conserved RecA-like or AAA+ folds that exist within a single subunit or between adjacent subunits that include “Walker A and B motifs” for ATP binding and hydrolysis and a basic “arginine finger” residue for nucleotide turnover and conformational coupling3,19–22. Conserved β-hairpin structures in AAA+ helicases contribute differentially to DNA binding and unwinding to direct DNA through the channel11,16,22–26, although additional interactions with DNA have also been detected on the exterior surface of helicases15,27,28.\n\nSeveral high-resolution X-ray and electron microscopy (EM) structures have been reported for the apo and nucleotide-bound forms of hexameric helicases (T7 gp429,30, DnaB31,32, Mito Twinkle33, SV40 Large-T11,34, Sulfolobus solfataricus MCM [SsoMCM]23,35, and Saccharomyces cerevisiae MCM [ScMCM2-7]36). The global shared architecture of the ring-shaped helicases is generally composed of two tiers: an N-terminal DNA-binding domain (NTD) and a C-terminal AAA+ or RecA motor domain (CTD) (Figure 1). The orientation of most helicases on DNA places the CTD toward the duplex double-stranded DNA (dsDNA) region and the NTD outwards22,37. The exception is E1, where the orientation is reversed, placing the NTD toward the duplex38. Thus, motor domains are often positioned close to the dsDNA duplex, which can leave the NTD regions free to bind, stabilize, or act on the resultant ssDNA.\n\nAcross species, hexameric helicase NTDs seem to have evolved differential functions. T7 gp4 can be expressed as either a 56 kDa helicase only form or as a full-length two-domain 65 kDa helicase-primase39. The composition of the T7 gp4 helicase hexamer is thought to be a mixture of the two forms in vivo, controlling the number of primases present for faster replication and less pausing. Other SF4 helicases, including T4 gp41 and Escherichia coli DnaB, interact with a separately encoded primase at the NTD in an analogous configuration. In those cases, the composition and ratio of helicase to primase is <1:1, and more often recognized as 6:331,40. For DnaB, ATP binding by the motor domain can induce conformational changes within the NTD collar that can regulate partner protein (i.e. DnaC or DnaG) selection41. As can be seen in Figure 1, increasing organismal complexity through the SF3 and SF6 helicases (from left to right) generally increases the size of the NTD to where they have evolved additional β-hairpins and zinc-finger motifs for more stabilized binding of the encircled strand and double hexamer formation42,43. The expanded NTD also provides a platform for control of activity through helicase accessory protein binding (in the case of Cdc45 and GINS44) or activation through phosphorylation by either cyclin-dependent kinase (CDK) or Dbf4-dependent Cdc7 kinase (DDK)45–47.\n\n\nHelicase loading and the encircling of DNA\n\nThe loading of hexameric helicases at replication origins and the associated steps required for encircling only one strand have been the subject of much debate over the years. What is clear is that the loading of the hexameric helicase generally requires the concerted action of accessory initiator proteins to locally melt duplex DNA and facilitate encircling of DNA. However, phage T7, mitochondrial Twinkle, and SV40 L-Tag helicases can load onto circular dsDNA on their own48–50. Within the three domains of life, the core ATPase activity and ordered assembly of replication initiation factors seem to be preserved to control the start of DNA synthesis51,52 (Table 1). In bacteria, the initiator, DnaA, forms a multimeric right-hand filament at the replication origin, oriC, to induce unwinding or melting at an A-T rich DNA unwinding element (DUE) (Figure 2A)53–60. Afterwards, the DnaB helicase is loaded on the top and bottom strands by concerted activities of DnaC and DnaA61–66. Once loaded, the primase, DnaG, interacts with DnaB, displaces DnaC40,67, and activates unwinding68. The association of an accessory helicase, Rep, with DnaB may aid in replication fork progression69–71.\n\nA) Loading of the bacterial DnaB helicase by the loader, DnaC, requires the destabilization of a DNA unwinding element (DUE) by the initiator protein DnaA. DnaB complexes with the primase, DnaG, to translocate along the lagging strand unwinding DNA ahead of the replication fork. B) Loading of the eukaryotic MCM2-7/Cdt1 complex requires the initial binding of the ORC complex (ORC1-6) and Cdc6. Interactions with Cdc6 and ORC with the CTD of MCM2-7 directs the adjacent loading of the first hexamer and dissociation of Cdc6 and Cdt1. Subsequent loading of the second hexamer is thought to proceed through direct interactions between the NTD of MCM2-7 to form the double hexamer. Activation of the helicase includes CDK phosphorylation of Sld2 and Sld3 to promote interaction with Dpb11 and stimulate DDK phosphorylation of MCM2/4/6 and recruitment of GINS and Cdc45 to form the CMG complex. Opening of the CMG complex and exclusion of the nontranslocating strand from the central channel activates unwinding and translocation on the leading strand. Gray and black boxes represent major and the foremost, respectively, queries remaining regarding structural conversions of helicases at origins.\n\nIn archaea and eukaryotes, the binding of the origin recognition complex (ORC1-6) and Cdc6 to origins of replication is necessary for loading MCM2-7/Cdt1 complexes onto dsDNA to generate a pre-replication complex (Pre-RC) (Figure 2B)72–76. The precise structural conformations and dynamics of MCM loading are not fully known, but the steps and components for assembly and activation of the eukaryotic MCM2–7 complex have been recently biochemically reconstituted in vitro, providing significant insight into the process77,78. In archaea, the homohexameric MCM complex exists as a closed ring in solution and would require initiation factors to stimulate opening into a helical conformation onto DNA79. Alternatively, increases in temperature for these model hyperthermophilic archaeal MCMs may provide the thermal energy required for the destabilization of a subunit interface required for loading. The eukaryotic MCM2-7 helicase appears to be naturally open, with a labile 2-5 interface that can be trapped by ORC/Cdc680–82. The ORC1-6 complex is arranged in a two-layered cracked ring that encircles DNA and uses the helix-turn-helix domains to engage the MCM2-7 hexamer in a proposed ring-ring interaction83,84, in a manner similar to the loading mechanism of clamp/clamp-loader complexes onto dsDNA85. The organization of the ORC complex also appears to be regulated and exists in either an autoinhibited ATP-bound form that precludes DNA binding or a proposed active form that requires a large conformational change in ORC1 that makes the complex competent for encircling DNA83. Afterwards, the first MCM2-7 hexamer is loaded through direct interactions of MCM6-Cdt1 with the ORC1-6/Cdc6 complex86,87. The second MCM2-7 hexamer is loaded through contacts between the NTDs of the first loaded MCM2-7 hexamer, rather than through interactions with the ORC1-6/Cdc6 complex78,84,88,89. This generated the double hexamer complex, which has been known for years for MCM (and SV40 L-Tag)42,90–92, but whether the double hexamer represents an active unwinding unit or an intermediate in the loading process was not known.\n\nAfter loading of the MCMs, a series of steps are required to form the active unwinding complex (Figure 2B). ATP hydrolysis by Cdc6 and ORC1 causes the dissociation of Cdc6 and Cdt193. Subsequent phosphorylation of Sld2 and Sld3 by CDK promotes Dpb11 (DNA polymerase B-associated protein) to interact with MCM2-794–96. Phosphorylation of Sld3, in particular, recruits Cdc45 and GINS (Sld5, Psf1, Psf2, Psf3) to MCM2-7 and stimulates the DDK-dependent phosphorylation of MCM297, as well as MCM4/698,99. These phosphorylation events allow opening of the MCM2/5 interface to extrude ssDNA that remains bound to Sld2/Sld3/Dpb11100,101. The active unwinding CMG complex or “unwindosome” is formed through the association of Cdc45 and GINS with MCM2-7 at the labile MCM2-MCM5 interface80,81 along the waist between the NTD and the CTD44,80,102. Cdc45 in particular blocks the MCM2-5 gate and prevents the loss of DNA from the central channel103. In the reverse mechanism, Cdc45 may also be important for converting MCM2-7 encircled on dsDNA to encircling only a single DNA strand while excluding the other. Formation of the CMG complex widens the gap between the NTD and CTD at MCM2 and MCM5 while concomitantly narrowing the interface at the opposite MCM4 and MCM6 subunits. This induced spiral configuration may contribute to coupled ATP hydrolysis, propagating a conformational change through the MCM2-7 complex to translocate along and unwind duplex DNA44,103.\n\n\nInteractions with DNA: views of the encircled strand\n\nX-ray structures of hexameric helicases with oligonucleotides bound in the central channel (E1, E. coli Rho [EcRho], Bacillus stearothermophilus [Bst] DnaB, and Pyrococcus furiosus MCM [PfuMCM]) have informed our understanding of the contacts and conformations required for translocation along ssDNA. In these co-crystal structures, ssDNA is bound in a helical conformation in the central channel making direct contacts with each subunit (Figure 3). For E1 and Rho, the hexameric ring is proposed to remain closed, but conformational changes between subunits, coupled with sequential ATP binding and hydrolysis around the ring, direct ssDNA through the central channel through contact with DNA binding loops in a staircase motion13,38,104. Each hexamer subunit interacts with one nucleotide of the oligo, predicating a step-size of one nucleotide per ATP hydrolyzed. This is consistent with the measured step-size of T7 gp4 of one base-pair unwound per dTTP hydrolyzed105. For DnaB, the crystal structure resembles more of a lock washer, where similar conformational changes throughout the quaternary structure facilitate movement, with a step-size of two nucleotides per ATP hydrolyzed, maintaining a cracked ring structure106. The ssDNA bound to the archaeal MCM seems to be trapped in a lateral orientation around the interior of the NTD, possibly identifying specific contacts during activation or unwinding, implying a step-size greater than one nucleotide per ATP hydrolyzed during translocation107. The EM structure of the intact eukaryotic CMG complex bound to DNA is in a spiral or lock washer conformation44, more similar to the DnaB/ssDNA complex. The crack in the ring between the MCM2 and 5 subunits is again held in check by the Cdc45 and GINS subunits and helps to stabilize the spiral configuration (Figure 3). Of course, the impact and absolute degree of spiraling, wrapping, or compaction of the encircled strand will need to be validated experimentally, most likely using single-molecule approaches to measure end-to-end distances during loading and unwinding. Almost certainly the flat ring, the asymmetrical spiral, and the cracked lock washer structures represent intermediates during helicase activation and unwinding, but both conformations will also need to be validated further with additional high-resolution structural studies or rigorous biophysical characterizations to monitor the changes in the conformations.\n\nSpace-filling representation of the C-terminal domain (E1 [SF3, 3’-5’] and DnaB [SF4, 5’-3’]) or N-terminal domain (Sso-Pfu hybrid [SF6, 3’-5’]) interacting with the encircled strand (purple). Also shown is the electron microscopy structure of the Drosophila Cdc45/MCM2-7/GINS complex (CMG) (SF6, 3’-5’) with color-coded subunits. The conformational states of the active translocating hexamers representing rings with subtle rises (E1) or obvious spirals (DnaB and CMG) in the structures as well as helical single-stranded DNA (ssDNA) are indicated in the schematics. The flat Sso-Pfu hybrid structure represents a nontranslocating state used to identify a novel lateral DNA binding site. The orange box arrow indicates the translocation direction of the hexamer relative to the encircled ssDNA.\n\n\nInteractions with DNA: Impact of the excluded strand\n\nBased on the structures and associated biochemical data, the steric exclusion (SE) model, where one strand is encircled and the other is physically excluded, has become the consensus opinion for unwinding for hexameric replication helicases (Figure 4)108. One caveat to this model is that it generally ignores any contributions of the excluded strand to unwinding. Electrostatic interactions with the excluded strand on the external surface of hexameric helicases have been noted for archaeal MCM and shown to be important for unwinding, contributing to the development of the steric exclusion and wrapping (SEW) model (Figure 4)17,109. Others have also noted that both ssDNA and dsDNA have a binding site on the external surface of other helicases15,27,28. The dynamic and somewhat stochastic nature of unwinding has been attributed to interactions of ssDNA on the external surface of hexameric helicases E138, T4 gp41110, and DnaB111. In addition, subunits within the unwindosome complexes of SV40 L-Tag112 and CMG113 have been shown to interact with the excluded strand for loading and activation of unwinding. Intriguingly, DNA repair helicases have also been shown to sense damage or modifications on the excluded strand and stall unwinding114–117.\n\nSteric exclusion (SE) model encircles the translocating strand and physically separates the nontranslocating strand outside of the central channel. The steric exclusion and wrapping (SEW) model takes into account specific interactions within the central channel (compaction or spiraling) as well as external interactions (binding or wrapping) with the excluded strand. Blue arrows indicate the direction of movement of the DNA strands with respect to the hexamer.\n\nFor SV40 L-Tag, initial binding to the origin may be directed by internal β-hairpins making direct contacts with the minor groove49 and specific contacts of the origin binding domain (OBD) to the major groove118. It is not currently understood how this initial dimer contact nucleates assembly of a double hexamer around dsDNA. Once loaded, SV40 L-Tag is proposed to convert from encircling duplex DNA to encircling ssDNA by pumping and extruding one strand out through side-channels11,119. Conformational changes within internal β-hairpins may direct the translocating strand through the central channel, while extruding the opposing strand. Using single-molecule experiments, researchers have shown that DNA unwinding proceeds with a single hexamer of L-Tag in a steric exclusion mechanism that is somewhat conformational mobile and able to bypass bulky adducts during translocation120. In comparison, a novel mechanism has recently been proposed for E1 where duplex DNA enters the hexamer before being separated internally and forcing individual strands out through separate exits channels121. Of course, the steps and dynamics for how these hexameric helicases convert from encircling duplex DNA to single strand separases by pumping DNA out through side-channels, opening of a gate, or through another unknown mechanism need to be visualized directly with high resolution. A recent EM structure shows the leading strand Pol ε ahead of the yeast CMG complex (at the CTD) and suggests a possible model where the encircled leading strand bends back and threads through a side-channel via the MCM2-5/Cdc45/GINS gate to enter the polymerase active site122. Alternative models of replisome-DNA interactions were also proposed in this study.\n\nWith this emerging information, excluded or opposing strand interactions shown in the SEW model (Figure 4) are poised to play multifaceted roles in loading, encircling, unwinding, and sensing of DNA. In the case of archaeal MCM, the external ssDNA binding path in the SEW model serpentines along the lateral length of the homohexamer, spanning the CTD and NTD, and even crossing and wrapping across multiple subunits (Graham & Trakselis, unpublished data)17. The SEW model for archaeal MCM is analogous to a socket wrench, whereby the encircling of one strand represents the socket and external interactions with the excluded strand represent the directional ratchet controlling the speed and stabilization of unwinding. Whether the SEW model is conserved in all or most hexameric helicases and/or at what stages of helicase assembly it may occur remains to be determined. Currently, we have found that external interactions and dynamics with the excluded strand in the E. coli DnaB helicase are practically identical to that of SsoMCM, despite their opposing polarities (Carney & Trakselis, unpublished). On the other hand, for T7 gp4 the excluded ssDNA interacts with T7 DNA polymerase to generate a replisome complex, where the helicase and polymerases are within one nucleotide of the fork junction and the helicase can make no external contact with the excluded strand105,123. Next, it will be important to determine which of the eukaryotic MCM subunits (MCM2–7) interact specifically with the excluded strand or whether uniform binding sites have evolved on all subunits. It is intriguing that this external contact may help fill in some of the missing steps highlighted in the gray or black boxes depicted in Figure 2.\n\nAlthough the structural features of the SEW model may be conserved with various hexameric helicases, both the mechanistic roles and molecular interaction sites may be different. In the case of SsoMCM, disruption of external interactions through mutagenesis reduced unwinding efficiency (3’-5’)17, but analogous external mutations on DnaB show a stimulation in unwinding (5’-3’) (Carney & Trakselis, unpublished). Modification of the excluded strand to a morpholino oligo similarly stimulates the unwinding rate of T7 gp4124. Whether these effects result from opposite unwinding polarities or finely tuned control of unwinding rates and maintenance of the excluded strand requires further testing. However, detection and identification of these novel external interactions may provide a unique opportunity to target specific helicases for inhibition. As none of the different hexameric helicase families exhibit significant sequence homology outside of the center P-loop NTPase fold, novel exterior patches (e.g. between the CTD and the NTD) may provide idealized locations for specific targeting of small molecules that perturb unwinding through disruption of excluded strand contacts and avoid direct inhibition of the internal conserved ATPase site.\n\n\nFuture directions\n\nAlthough significant advances in our understanding of hexameric helicase assembly, loading, and unwinding have been made over the past few years from quantitative biophysical characterizations and various high-resolution structures, more work is required to reveal specific mechanistic steps and transitions. For example, the essential components for the initial loading of hexameric helicases onto DNA are well described, but the conformational changes that occur within the hexamers during the encircling of ssDNA are still unknown. After all these years, the black box in the whole mechanism is still the structural conversion of the helicase from encircling dsDNA to the encircled ssDNA directing the polarity of translocation and unwinding, primarily for SF3 and SF6 enzymes. Although much is known about the loading and activation mechanism in the Gram-negative E. coli system, far less is known about SF4 helicases in the Gram-positive organisms where DnaI acts as the loader65,125,126 or in bacteria which lack DnaC/DnaI loader homologs altogether127,128.\n\nAlthough there is a wealth of structural information on the static hexameric helicases themselves, there is still much debate on the mechanics of helicase action. No longer is the focus directly on the structure of the helicase protein itself. Instead, it has switched from identifying conformational changes, transacting proteins, and post-translational modifications that reveal how duplex DNA is destabilized and the path it takes to be excluded. Finally, although the unwinding mechanism of hexameric helicases was thought to be as simple as excluding one strand from the central channel, new information highlighting the specificity and importance of interactions with the nontranslocating strand have central implications on loading and unwinding mechanisms. It is these dynamic conformational steps from the viewpoint of both the helicase and the duplex DNA that will lead to the next transformational leap in replication helicase discovery.\n\n\nAbbreviations\n\nAAA+, ATPases associated with a variety of activities; ATP, adenosine triphosphate; Cdc6, cell division cycle 6; Cdc45, cell division cycle 45; CDK, cyclin-dependent kinase; Cdt1, chromatin licensing and DNA replication factor 1; CMG, Cdc45/MCM2-7/GINS complex; CTD, C-terminal domain; DDK, Dbf4-dependent Cdc7 kinase; Dpb11, DNA polymerase B-associated protein; dsDNA, double-stranded DNA; EM, electron microscopy; GINS, go-ichi-nii-san, Japanese for 5-1-2-3 for Sld5-Psf1-Psf2-Psf3; MCM, minichromosome maintenance proteins; NTD, N-terminal domain; NTPs, nucleotide triphosphates; ORC, origin recognition complex; Pre-RC, prereplication complex; Psf, partner with Sld5; SE, steric exclusion; SEW, steric exclusion and wrapping; SF, superfamily; Sld, synthetic lethal with dpb11; ssDNA, single-stranded DNA.", "appendix": "Competing interests\n\n\n\nThe author declares that he has no disclosures.\n\n\nGrant information\n\nResearch in the Trakselis laboratory is sponsored by the American Cancer Society (RSG-11-049-01-DMC) and Baylor University.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nI am grateful to the broader helicase field as well as individual members of my laboratory (both past and present) for their inspiration, discussions, and collegiality. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "11998", "date": "27 Jan 2016", "name": "Kevin Raney", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "11999", "date": "27 Jan 2016", "name": "James Berger", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12000", "date": "27 Jan 2016", "name": "Smita Patel", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-111
https://f1000research.com/articles/5-110/v1	27 Jan 16	{ "type": "Review", "title": "Recent advances in understanding Type 1 Diabetes", "authors": [ "Gustaf Christoffersson", "Teresa Rodriguez-Calvo", "Matthias von Herrath", "Gustaf Christoffersson", "Teresa Rodriguez-Calvo" ], "abstract": "Type 1 diabetes is a multifactorial disease in which genetic and environmental factors play a key role. The triggering event is still obscure, and so are many of the immune events that follow. In this brief review, we discuss the possible role of potential environmental factors and which triggers are believed to have a role in the disease. In addition, as the disease evolves, beta cells are lost and this occurs in a very heterogeneous fashion. Our knowledge of how beta cell mass declines and our view of the disease’s pathogenesis are also debated. We highlight the major hallmarks of disease, among which are MHC-I (major histocompatibility complex class I) expression and insulitis. The dependence versus independence of antigen for the immune infiltrate is also discussed, as both the influence from bystander T cells and the formation of neo-epitopes through post-translational modifications are thought to influence the course of the disease. As human studies are proliferating, our understanding of the disease’s pathogenesis will increase exponentially. This article aims to shed light on some of the burning questions in type 1 diabetes research.", "keywords": [ "Type 1 Diabetes", "Beta Cells", "Environmental Triggers", "Beta Cell Mass", "MHC-I expression", "Insulitis" ], "content": "Introduction\n\nFor many years, type 1 diabetes (T1D) was defined as an autoimmune disease in which autoreactive T cells escape negative selection and destroy the insulin-producing beta cells. Nowadays, we know that the disease is complex, involving many immune and non-immune elements, many of which remain obscure. Several potential environmental triggers of the disease have been described, with viral infections at the top of the list. Alas, still no definitive evidence has been found that directly proves them to be the causative agents of T1D.\n\nRecent access to pancreatic tissue from T1D subjects (thanks to the efforts of several biobanks around the world) has shown great variability in beta cell mass decline in patients on their first year post-diagnosis, arguing for the existence of an unknown amount of remaining beta cells in the pancreas. Moreover, insulitis, one of the hallmarks of T1D, has been shown to be a heterogeneous lesion in humans, suggesting that the immune attack is not orchestrated entirely by T cells, as the pace of destruction is different for every individual. The innate immune system and non-specific inflammation have been increasingly linked to T1D and are contributing factors to the pathogenesis of this complex disease. Thus, T1D might not be a fully T cell-mediated disease, as it seems not to be entirely antigen driven. Our knowledge of the abundance and role of antigen-specific T cells is very limited. T cells autoreactive against known beta cell antigens are very scarce in both peripheral blood and the pancreas of patients with T1D. This suggests that there are other non-antigen-specific cells involved or that we do not know the full specificities of the immune infiltrate. In this regard, efforts are being made by the scientific community to discover new autoantigens in T1D, among which are possible post-translational modifications (PTMs) that lead to the formation of new antigens not previously recognized by the immune system. In this brief review, we navigate through the pathogenesis of T1D, from possible triggers to classic models and dogmas of the disease, summarizing recent findings in prioritized areas of the field.\n\n\nViral infection and other possible triggers: how do we deal with the environment?\n\nT1D is an inherently difficult disease to study. The main destructive autoimmune events in the pancreas and the trigger of these may pre-date diagnosis by several years. This discrepancy in time between disease triggering and diagnosis makes it very difficult to draw conclusions about the cause of the disease from samples taken from patients with T1D; it’s like painting a portrait of a person you barely see the shadow of.\n\nOver the years, epidemiological studies have shown a significant correlation between the environment and the development of T1D: there is currently a 3% annual increase in incidence, which cannot be explained by genetic predisposition (e.g. MHC [major histocompatibility complex] class II haplotypes)1,2, there is high heterogeneity in the geographical distribution of the disease3, and, in the offspring of immigrants, T1D incidence rate will be approximately that of their new country of residence4.\n\nA wide range of environmental factors have been shown to correlate with T1D development in susceptible individuals, such as dietary factors (e.g. cow’s milk and gluten5,6), toxins (e.g. N-nitroso compounds7), stress8, and enteroviral infection (e.g. coxsackievirus B9). The composition of the gut microbiome is emerging as another major player in autoimmunity. Several animal studies indicate that alterations in the intestinal microbiota are associated with the development of autoimmune diabetes. In bio-breeding rats, a difference in microbiota composition was seen prior to onset of T1D in animals that are prone to the disease versus ones that are protected10, and a transferable protection has also been observed in non-obese diabetic mice lacking MyD88 (myeloid differentiation primary response gene 88), an essential signal transducer in Toll-like receptor signaling11,12. Studies showing an influence of the human microbiome and the development of T1D have been few and limited in numbers of study subjects13. Recently, however, a study using longitudinal data from 33 Finnish and Estonian children reports that a shift in microbiota occurred after seroconversion but before onset of T1D14. Still, a direct relationship between the gut and the development of T1D in humans remains to be established. Nonetheless, these recent findings prompt further investigation into the correlation of the microbiome and the increased gut permeability seen in T1D subjects15 and its prospective contribution to the entry of possible T1D-promoting agents such as enterovirus.\n\nDespite the plethora of studies that have implicated virus as a major environmental factor in the development of T1D, evidence remains indirect. A recent report took a proteomics approach to assess the anti-viral response in 42 young patients with T1D16. Even though the array of viruses was not exhaustive, only one strain, Epstein-Barr virus, correlated with recent-onset T1D. In an effort to attain highly relevant tissue for the main purpose of investigating viral influence in disease onset, pancreas tail resections from patients with recent-onset (3 to 9 weeks after diagnosis) T1D were collected as part of the Diabetes Virus Detection (DiViD) study17. In these samples, evidence for an ongoing enteroviral infection was detected in all six patients through the expression of enteroviral capsid protein 1 (VP1) in some of the remaining islets18. Causality has yet to be proven in the case of viral infection, but recent advances in molecular techniques and access to highly relevant tissues (as illustrated above) might help close the temporal gap between the trigger of the disease and diagnosis.\n\n\nWhat does the type 1 diabetes scenario look like nowadays? The relapsing-remitting model\n\nIn 1986, George S. Eisenbarth proposed that T1D was a “genetically programmed autoimmune disease”19. In his view, six different stages could be distinguished: (I) genetic susceptibility, (II) triggering event, (III) active autoimmunity, (IV) immune abnormalities accompanied by loss of glucose-stimulated insulin secretion, (V) overt diabetes with few remaining beta cells, and (VI) complete loss of beta cells. This model was presented in parallel with the stages of beta cell destruction; normal beta cell mass remained in stage I, whereas in stage V, at onset, only 10% of beta cell mass was left. At stage VI, a complete loss was described. Nonetheless, it was one of the first attempts to explain the pathogenesis of T1D and beta cell decline that provided a useful framework to conduct clinical research studies and was widely accepted at the time. The linear beta cell mass decline model was adopted for many years; this was due in part to the lack of proper tools to find a good correlate for remaining beta cell mass and the absence of longitudinal studies capable of following patients during the pre-diabetic phase and after diabetes onset.\n\nThere has been some debate as to whether or not the linearity of Eisenbarth’s model was accurate. Other models depicting variable beta cell mass decay have been described20. Accordingly, these models have tried to include several scenarios in which genetic predisposition and environmental triggers might play different roles21. Among these, the relapsing-remitting hypothesis has been well accepted as an alternative scenario in the disease’s pathogenesis22. In this model, a fluctuation in beta cell mass is present over time as a consequence of different waves of beta cell killing in a complex battle among regulatory elements, autoreactive cells, beta cell survival, and their subsequent feedback systems.\n\nAfter three decades of contemplating the idea of a linear, variable, or relapsing-remitting model, or a combination of these, it has become more evident that these models might be partially right or partially wrong, depending on which stage of the disease we are looking at. A recent study by Herold et al.23 showed the presence of elevated levels of unmethylated insulin (INS) DNA and their correlation with beta cell death in a cohort of at-risk patients. Beta cell killing starts long before diabetes onset, but interestingly the increase in beta cell death is subtle and sporadic prior to diagnosis, with only a mild repercussion on insulin secretion. However, a marked increase in unmethylated INS DNA is seen closer to the time of diagnosis, declining after the development of hyperglycemia. This would suggest that both the relapsing-remitting and the linear models could be applied to the pre-diabetes and peri-diagnosis periods, respectively, with small cyclic waves of beta cell destruction followed by an abrupt fall of beta cell mass at diagnosis. This is in agreement with previous publications in which beta cell function seems stable for many years, declining only around the time of diagnosis24–26. In accordance, a recent publication by Fisher et al.27 revealed an increase in beta cell death at onset, which diminished at 8 weeks post-diagnosis, a phenomenon that appeared to be specific to the onset of T1D. In chronic infections, the persistent exposure to antigen is associated with an exhaustion of CD8+ T cells and the failure to clear the virus28. Similarly, CD8+ T cell exhaustion has also been observed in autoimmunity (including T1D), in part explaining the relapsing-remitting nature of these diseases. After a while, the immune system can revert the exhaustion and again have fully functional autoreactive CD8+ T cells29 capable of continuing their attack on the beta cells. Ultimately, a critical amount of beta cell mass is lost and the death of beta cells cannot be compensated, leading to the development of hyperglycemic symptoms and the rapid beta cell demise around the time of diagnosis.\n\nSeveral scenarios of beta cell decline have been described in the past; however, the ideal model would also take into account clinical features of the disease. In an attempt to do so, the Juvenile Diabetes Research Foundation (JDRF), the Endocrine Society, and the American Diabetes Association recently suggested the adoption of a new “Staging Classification System” for T1D that also integrates clinical aspects of disease progression. The disease is divided into three phases: stage 1: autoimmunity and/or normoglycemia or presymptomatic T1D; stage 2: autoimmunity and/or dysglycemia or presymptomatic T1D; and stage 3: autoimmunity and/or dysglycemia or symptomatic T1D. In these stages, there is a variable progression from beta cell autoimmunity to glucose intolerance and to the symptomatic stage30. Whichever model is chosen to stratify the different phases of the disease, the means of detecting beta cell destruction and survival should be further investigated in order to evaluate their potential as biomarkers and design better therapeutic strategies adapted to the patient’s needs and according to the number of beta cells present in the pancreas at diagnosis.\n\n\nRemaining beta cell mass at onset and after diagnosis: beta cell destruction versus preservation\n\nIn the model described by Eisenbarth, only around 10% of beta cell mass remained at diagnosis19. As many have discussed before, “diagnosis” might have different meanings depending on the time of disease progression. By definition, diagnosis is the “identification of the nature of an illness or other problem by examination of the symptoms”. Therefore, the keys are the stage at which disease progression symptoms appear and whether the patient is sufficiently aware of them. Consequently, every patient might be diagnosed at a different time point in the disease course31. In addition, many patients still produce small amounts of C-peptide, especially if they are on their first 2 years post-diagnosis31. An important study by Oram et al. showed that 80% of people with T1D for at least 5 years had detectable endogenous C-peptide, supporting the notion that after diagnosis an unknown number of beta cells remain in the pancreas32. In accordance, no changes to the 2-hour Oral Glucose Tolerance Test (OGTT) were detected until 0.8 years prior to diagnosis in the Diabetes Prevention Trial-Type 1 (DPT-1), followed by a rapid decline in the OGTT response post-diagnosis26. A decrease in stimulated C-peptide was also observed 6 months prior to the appearance of the first symptoms, with levels rapidly declining 3 months before the symptomatic phase24. Moreover, C-peptide decline does not seem to occur at the same rate in adults versus children, the latter showing a 50% decline in the first year, whereas in the former the 1-year decline is set at 20%33. Overall, different disease progression to diagnosis coupled with the heterogeneity of the disease makes it difficult to establish a standard for the number of remaining beta cells at diagnosis.\n\nThe access to human pancreatic tissue samples has been critical to estimate the number of residual beta cells in T1D donors34,35. Studies emanating from the JDRF Network for Pancreatic Organ donors with Diabetes (nPOD) have shown that beta cell mass is very heterogeneous, even in non-diabetic individuals34. In addition, recent reports have shown that a small number of patients with T1D still have remaining insulin-producing cells many years after diagnosis36,37, and this probably explains the presence of detectable C-peptide in some of these patients. Three interesting scenarios can be contemplated in light of recent findings: (1) several waves of beta cell destruction in a relapsing-remitting fashion over the pre-diabetic period and normal C-peptide production, (2) an abrupt fall in beta cell mass at diagnosis and abnormally low C-peptide levels, and (3) possible formation of new beta cells or proliferation during the diabetic phase (enough to produce a small amount of insulin and allow C-peptide detection) (Figure 1).\n\nGenetically predisposed individuals are exposed to a triggering event, which induces inflammation and upregulation of major histocompatibility complex class I (MHC-I) molecules in endocrine cells. Autoantibodies against islet antigens appear in the circulation during the autoantibody-positive phase. In this period, blood glucose levels are normal and there are no evident symptoms of the disease. During this chimera of normality, autoreactive T cells proliferate and start to infiltrate the islets, causing waves of beta cell death. In this period, an imbalance between beta cell death and survival generates small fluctuations in beta cell mass. The immune infiltration becomes apparent during the pre-diabetic phase, in which infiltrating cells actively destroy beta cells at a faster pace, a phenomenon that cannot be overcome by endogenous regulatory elements. Dysregulation of glucose levels starts to appear because of the inability of the remaining beta cells to produce enough insulin. In addition, post-translational modifications (PTMs) might arise as a consequence of endoplasmic reticulum stress and inflammation, potentiating the activation of cytotoxic CD8+ T cells and further beta cell destruction. PTMs might expand into the diabetic period, as inflammation is still present. At diagnosis, the number of cells infiltrating the islets is large and insulitis becomes more apparent. The decline in beta cell mass makes it impossible to control glucose levels, and the first symptoms of disease start to appear. During the first years of the diabetic period, C-peptide might still be detectable as some beta cells manage to survive the immune attack. However, as beta cell mass diminishes, so do inflammation and infiltration. Overall, fluctuations in beta cell mass over the years create a continuous relapsing-remitting phenotype with a more abrupt fall around the time of diagnosis, when a critical beta cell mass is reached.\n\nAs pointed out by many, there is a need for more insights into the pathology of human T1D in order to understand the sequence of events that might take place in the pancreas before and after diagnosis: how and when are beta cells destroyed? How and when do immune cells attack the islets? The answers to these questions will have enormous repercussions for clinical intervention before and after diagnosis.\n\n\nHallmarks of the disease: insulitis, antigen specificity, MHC-I, and heterogeneity\n\nThe diabetes research community has relied heavily on animal models and post-mortem-donated human pancreatic tissue for their studies. Thus, most of what we know about the initial stages of autoimmune diabetes will be either snapshots from human pathology samples or data extrapolated from animal models. However, it is clear that the disease presents with autoantibodies before symptoms arise and that there is substantial islet inflammation (insulitis)38, where an autoreactive destruction of insulin-producing cells by CD8+ T cells is ongoing37.\n\nThe postulated presence of autoreactive CD8+ T cells in the islets of patients with T1D was proven by tetramer stainings of pancreatic sections37. What was evident in this study was that the number of CD8+ T cells at the islets could not be completely explained by their reactivity toward islet antigens. The majority of CD8+ T cells in the insulitic lesion are not islet specific, and the proportion of specific cells could be as low as 1%39. These observations are likely the result of two factors: (1) all autoreactive antigens have not been mapped yet, and this is due in part to PTM (discussed below), and (2) the ongoing inflammation in the islets likely recruits non-specific bystanders by factors such as chemokines expressed by lymphocytes and myeloid cells present at this site40,41. The importance of the specificity versus non-specificity of the T cells at this site is not clear at the moment, but the general hypothesis is that additive damage to the insulin-producing cells occurs via the non-specific release of beta cell-toxic cytokines.\n\nAs mentioned above, the majority of CD8+ T cells present in the insulitic lesion are not reactive against “known” beta cell antigens. T cells recognizing autoantigens in the thymus and bone marrow are deleted to avoid recognition of self-peptides in tissues and, consequently, autoimmunity. However, central tolerance does not account for potential PTMs that might arise in life. PTMs alter the primary sequence of the protein, which has a potential impact on MHC-peptide affinity or MHC–peptide–T cell receptor interactions42,43. PTMs can be mediated by enzymes like peptidyl arginine deiminase (PAD) or tissue transglutaminase 2 (TG2) or occur spontaneously44; they can cause modifications of protein structure or their biological function or cause degradation. The activity of modifying enzymes can be enhanced by endoplasmic reticulum (ER) stress or other environmental triggers, among them viral or bacterial infections. Alternatively, beta cell antigens could also trigger the first autoimmune attack, in turn activating modifying enzymes and ultimately propagating autoreactivity, epitope spreading, and the perpetuation of the autoimmune attack. As part of the immune response, modified antigens could be processed and presented by antigen-presenting cells to T cells and B cells.\n\nThe first evidence for PTM occurring in T1D was the observation of T cell reactivity toward an altered insulin epitope where a non-native disulfide bond had formed through spontaneous oxidation45. Since then, both PAD-meditated citrullination46 and TG2-mediated transglutamination47 of islet antigens have been found in patients with T1D. For example, one of the major autoantigens, GAD65, can be found in both transglutaminated and citrullinated forms, thus being modified by both major PTM enzymes48. The high metabolic rate in beta cells makes them highly susceptible to ER stress49,50. This, combined with the islet inflammation in T1D, leads to a high probability of PTMs occurring. Thus, PTMs of islet antigens could be a major factor in the non-linearity of T1D progression (discussed above), as the self-propagation of the inflammation and cellular stress could lead to the genesis of neo-antigens that ultimately would increase the rate of autoimmune beta cell destruction.\n\nAnother histopathologic hallmark of T1D besides the lymphocyte infiltrate in the islets is the hyperexpression of MHC-I on endocrine cells. Since the first observation in the 1980s, islets from T1D subjects have been found to overexpress MHC-I and interferon-α in several studies51,52. However, the only evidence for MHC-I overexpression is from immunostaining of tissue sections18,37,53. A recent article tried to find quantitative evidence for an increase in MHC-I at the mRNA and protein levels54. In this study, MHC-I hyperexpression was detected by immunohistochemical methods, but the limited comparisons performed failed to correlate with quantitative mRNA detection methods, questioning the relevance of previous observations. However, it is known that mRNA is rapidly degraded under the harsh conditions present in the pancreas. In addition, several laboratories have reported the presence of MHC-I hyperexpression in T1D donors by using different antibodies in different specimens preserved under different conditions (frozen versus formalin-fixed paraffin-embedded)18,37,53,55,56. This overexpression of MHC molecules and type I interferons is enigmatic but throws suspicion on a possible viral infection of the pancreas as a contributing factor to T1D. Common to both insulitis and MHC-I expression is heterogeneity in the distribution throughout the organ. The histopathologic features of the pancreas are lobular, and so large parts can be affected while some lobules remain undisturbed56,57. This asymmetrical anatomical distribution of the lesions could be a consequence of a viral infection or a feature of an anatomical distribution network such as the nervous58, vascular59–61, or acinar-ductal62 systems.\n\nThe key to understanding the initial autoimmune events in T1D will be to know why beta cells upregulate MHC molecules and what controls the initial lymphocytic infiltration of the islets. Is MHC-I hyperexpression just a pseudophenomenon in the wake of insulitis, or does it have a disease-propagating value? Is antigen a main gatekeeper for CD8+ T cells in the developing insulitic lesion, or are other, less appreciated signals (e.g. innate, neuronal, or vascular) at play?\n\n\nConclusions\n\nEnvironmental triggers continue to be enigmatic, but longitudinal studies that are following patients over many years could shed light on potential environmental influence if a sufficiently strong correlation with disease progression is found. The ambitious pancreas tissue biobank projects and novel biopsy studies have already, shortly after their inception, changed our view of the disease. They have allowed investigators around the world to look into the pancreas and have a glimpse of the residual beta cell mass in the pre-diabetic phase, at diagnosis, and many years thereafter. The evolution of the field during the last few decades has proven that our views of the disease’s cause and progression of inflammation are in constant need of reassessment. Further efforts should be made in order to identify potential new autoantigens and PTMs and characterize bystander inflammation in order to know what facilitates immune cell entry into the islets. In this short review, we aimed to highlight some of the major remaining questions in T1D research, areas that we believe may be key to solving this enigmatic disease.\n\n\nAbbreviations\n\nER, endoplasmic reticulum; JDRF, Juvenile Diabetes Research Foundation; MHC, major histocompatibility complex; OGTT, Oral Glucose Tolerance Test; PAD, peptidyl arginine deiminase; PTM, post-translational modification; T1D, type 1 diabetes; TG2, transglutaminase 2.", "appendix": "Competing interests\n\n\n\nMGvH is an employee of Novo Nordisk. The other authors declare that they have no competing interests.\n\n\nGrant information\n\nGC is supported by a postdoctoral fellowship from the Swedish Research Council.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nWe thank biobanks worldwide for continuously providing pancreas samples and allowing us to improve our knowledge of type 1 diabetes. We thank all the families and individuals who have generously donated organs for research purposes as well as those who participated or are involved in clinical trials. Their gifts to the scientific community are of incalculable value.\n\n\nReferences\n\nHarjutsalo V, Sjöberg L, Tuomilehto J: Time trends in the incidence of type 1 diabetes in Finnish children: a cohort study. Lancet. 2008; 371(9626): 1777–82. PubMed Abstract \| Publisher Full Text\n\nDIAMOND Project Group. 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PubMed Abstract \| Publisher Full Text\n\nMannering SI, Harrison LC, Williamson NA, et al.: The insulin A-chain epitope recognized by human T cells is posttranslationally modified. J Exp Med. 2005; 202(9): 1191–7. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nRondas D, Crèvecoeur I, D'Hertog W, et al.: Citrullinated glucose-regulated protein 78 is an autoantigen in type 1 diabetes. Diabetes. 2015; 64(2): 573–86. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nvan Lummel M, Duinkerken G, van Veelen PA, et al.: Posttranslational modification of HLA-DQ binding islet autoantigens in type 1 diabetes. Diabetes. 2014; 63(1): 237–47. PubMed Abstract \| Publisher Full Text\n\nMcGinty JW, Chow IT, Greenbaum C, et al.: Recognition of posttranslationally modified GAD65 epitopes in subjects with type 1 diabetes. Diabetes. 2014; 63(9): 3033–40. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nOrtsäter H, Sjöholm A: A busy cell--endoplasmic reticulum stress in the pancreatic beta-cell. Mol Cell Endocrinol. 2007; 277(1–2): 1–5. PubMed Abstract \| Publisher Full Text\n\nEizirik DL, Cardozo AK, Cnop M: The role for endoplasmic reticulum stress in diabetes mellitus. Endocr Rev. 2008; 29(1): 42–61. PubMed Abstract \| Publisher Full Text\n\nFoulis AK, Farquharson MA, Hardman R: Aberrant expression of class II major histocompatibility complex molecules by B cells and hyperexpression of class I major histocompatibility complex molecules by insulin containing islets in type 1 (insulin-dependent) diabetes mellitus. Diabetologia. 1987; 30(5): 333–43. PubMed Abstract \| Publisher Full Text\n\nFoulis AK, Farquharson MA, Meager A: Immunoreactive alpha-interferon in insulin-secreting beta cells in type 1 diabetes mellitus. Lancet. 1987; 2(8573): 1423–7. PubMed Abstract \| Publisher Full Text\n\nFoulis AK: C. L. Oakley lecture (1987). The pathogenesis of beta cell destruction in type I (insulin-dependent) diabetes mellitus. J Pathol. 1987; 152(3): 141–8. PubMed Abstract \| Publisher Full Text\n\nSkog O, Korsgren S, Wiberg A, et al.: Expression of human leukocyte antigen class I in endocrine and exocrine pancreatic tissue at onset of type 1 diabetes. Am J Pathol. 2015; 185(1): 129–38. PubMed Abstract \| Publisher Full Text\n\nRichardson SJ, Willcox A, Bone AJ, et al.: Immunopathology of the human pancreas in type-I diabetes. Semin Immunopathol. 2011; 33(1): 9–21. PubMed Abstract \| Publisher Full Text\n\nRodriguez-Calvo T, Suwandi JS, Amirian N, et al.: Heterogeneity and Lobularity of Pancreatic Pathology in Type 1 Diabetes during the Prediabetic Phase. J Histochem Cytochem. 2015; 63(8): 626–36. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGianani R, Campbell-Thompson M, Sarkar SA, et al.: Dimorphic histopathology of long-standing childhood-onset diabetes. Diabetologia. 2010; 53(4): 690–8. PubMed Abstract \| Publisher Full Text\n\nRazavi R, Chan Y, Afifiyan FN, et al.: TRPV1+ sensory neurons control beta cell stress and islet inflammation in autoimmune diabetes. Cell. 2006; 127(6): 1123–35. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nZanone MM, Favaro E, Ferioli E, et al.: Human pancreatic islet endothelial cells express coxsackievirus and adenovirus receptor and are activated by coxsackie B virus infection. FASEB J. 2007; 21(12): 3308–17. PubMed Abstract \| Publisher Full Text\n\nSavinov AY, Wong FS, Stonebraker AC, et al.: Presentation of antigen by endothelial cells and chemoattraction are required for homing of insulin-specific CD8+ T cells. J Exp Med. 2003; 197(5): 643–56. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChristoffersson G, Waldén T, Sandberg M, et al.: Matrix metalloproteinase-9 is essential for physiological Beta cell function and islet vascularization in adult mice. Am J Pathol. 2015; 185(4): 1094–103. PubMed Abstract \| Publisher Full Text\n\nKorsgren S, Molin Y, Salmela K, et al.: On the etiology of type 1 diabetes: a new animal model signifying a decisive role for bacteria eliciting an adverse innate immunity response. Am J Pathol. 2012; 181(5): 1735–48. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation" }	[ { "id": "12001", "date": "27 Jan 2016", "name": "Alberto Pugliese", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12002", "date": "27 Jan 2016", "name": "Li Wen", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-110
https://f1000research.com/articles/5-109/v1	27 Jan 16	{ "type": "Research Article", "title": "Associations between joint effusion in the knee and gene expression levels in the circulation: a meta-analysis", "authors": [ "Marjolein J. Peters", "Yolande F.M. Ramos", "Wouter den Hollander", "Dieuwke Schiphof", "Albert Hofman", "André G. Uitterlinden", "Edwin H.G. Oei", "P. Eline Slagboom", "Margreet Kloppenburg", "Johan L. Bloem", "Sita M.A. Bierma-Zeinstra", "Ingrid Meulenbelt", "Joyce B.J. van Meurs", "Marjolein J. Peters", "Wouter den Hollander", "Dieuwke Schiphof", "Albert Hofman", "André G. Uitterlinden", "Edwin H.G. Oei", "P. Eline Slagboom", "Margreet Kloppenburg", "Johan L. Bloem", "Sita M.A. Bierma-Zeinstra", "Ingrid Meulenbelt", "Joyce B.J. van Meurs" ], "abstract": "Objective: To identify molecular biomarkers for early knee osteoarthritis (OA), we examined whether joint effusion in the knee associated with different gene expression levels in the circulation.Materials and Methods: Joint effusion grades measured with magnetic resonance (MR) imaging and gene expression levels in blood were determined in women of the Rotterdam Study (N=135) and GARP (N=98). Associations were examined using linear regression analyses, adjusted for age, fasting status, RNA quality, technical batch effects, blood cell counts, and BMI. To investigate enriched pathways and protein-protein interactions, we used the DAVID and STRING webtools.Results: In a meta-analysis, we identified 257 probes mapping to 189 unique genes in blood that were nominally significantly associated with joint effusion grades in the knee. Several compelling genes were identified such as C1orf38 and NFATC1. Significantly enriched biological pathways were: response to stress, gene expression, negative regulation of intracellular signal transduction, and antigen processing and presentation of exogenous pathways.Conclusion: Meta-analyses and subsequent enriched biological pathways resulted in interesting candidate genes associated with joint effusion that require further characterization. Associations were not transcriptome-wide significant most likely due to limited power. Additional studies are required to replicate our findings in more samples, which will greatly help in understanding the pathophysiology of OA and its relation to inflammation, and may result in biomarkers urgently needed to diagnose OA at an early stage.", "keywords": [ "knee osteoarthritis", "joint effusion", "molecular markers", "inflammation", "blood" ], "content": "Introduction\n\nOsteoarthritis (OA) is a common, age-related, degenerative disease of the synovial joints. It is characterized by cartilage degradation, osteophyte formation, subchondral bone changes, and synovitis1. These characteristics can lead to joint space narrowing, pain, and loss of function, until at the end-stage of the disease total joint replacement is required. OA is a leading cause of morbidity and disability and carries high socioeconomic costs. With increasing obesity and age in the population, a massive rise in morbidity and costs attributed to OA is expected. To be able to change from symptomatic treatment at late disease state and total joint replacement towards early (secondary) prevention, it is very important to identify new osteoarthritic disease stage markers that could be measured in the early stages of OA. These markers should function as new targets or biomarkers for early disease treatment and prevention.\n\nRadiography is routinely used to support the diagnosis of OA. However, radiographic imaging is inadequate to detect and monitor biochemical changes within joint tissues which can occur long before symptoms are present. Magnetic resonance (MR) imaging is a non-invasive 3D imaging method with high tissue contrast that has been successfully used to visualize osteoarthritic changes2. In addition to radiographic osteophyte formation and joint space loss, joint effusion can be assessed. Joint effusion is the presence of increased intra-articular fluid3, which has been positively associated with knee pain in knee OA patients4. Joint effusion is known to be related to joint inflammation5 and a recent study showed that occurrence of joint effusion is a strong predictor for development of incident radiographic OA6.\n\nAs inflammation is increasingly considered to be an important pathway in the OA pathophysiology, efforts have been made to identify pro- and anti-inflammatory mediators (such as cytokines) which enable monitoring of the OA disease course7–9. With the aim to better understand the downstream consequences of inflammation in the knee, we compared gene expression levels in the blood of participants with different grades of joint effusion, as assessed by MR imaging. Ramos et al. already identified specific gene expression networks in blood associated with OA status10. Therefore, it could be advocated that blood expression profiles may reflect predisposition to OA. And because blood is a readily accessible tissue, gene expression levels associated with joint effusion may serve as molecular biomarkers for early detection of OA. We examined in two cohort studies whether joint effusion grades on MR imaging of the knee were associated with specific gene expression levels in the peripheral circulation, and subsequently performed a meta-analysis. Analysis for enrichment was performed to determine whether particular pathways were overrepresented among the genes associated with joint effusion.\n\n\nMaterials and methods\n\nThe Rotterdam Study (RS) is a large prospective, population-based cohort study in the district of Rotterdam, the Netherlands, investigating the prevalence, incidence, and risk factors of various chronic disabling diseases among elderly Caucasians aged 45 years and over. A detailed description of the design and rationale of the Rotterdam Study has been published elsewhere11. We invited the first 1,116 women aged 45–60 years visiting the research center to join a sub-study investigating early signs of knee osteoarthritis (knee OA). Participants were evaluated for the self-reported presence of rheumatoid arthritis (RA) and these cases were excluded. An additional exclusion criterion was the presence of any contra-indications for MR imaging, including weighing more than 150 kilograms. In total, 891 participants were included. For this study, we selected participants having both gene expression data and good quality knee MR imaging data available. In total, we could include 135 participants. The Rotterdam Study has been approved by the Medical Ethics Committee of the Erasmus MC and by the Ministry of Health, Welfare and Sport of the Netherlands, implementing the “Wet Bevolkingsonderzoek: ERGO (Population Studies Act: Rotterdam Study)”. All participants provided written informed consent to participate in the study and to obtain information from their treating physicians11.\n\nThe Genetics, Arthrosis and Progression study (GARP) consists of 191 sibling pairs (n=382) of white, Dutch ancestry. All participants (age range 40–78 years; mean age 60 years) are clinically and radiographically diagnosed with primary, symptomatic OA at multiple joint sites in the hand, or in at least two joints of the following locations: hand, spine (cervical or lumbar), knee, or hip12. Patients with secondary OA, such as inflammatory joint disease, major developmental diseases, bone dysplasia, major local factors or metabolic diseases as hemochromatosis were excluded. Sibling pairs (n=105) with at least one subject with symptomatic hip or knee OA (but not in a radiographic end-stage) were eligible for the MR imaging sub-study13; in 5 out of 210 patients no MR imaging (one due to claustrophobia, one with a large knee that did not fit into the knee coil) or an MR imaging of insufficient quality (due to motion artefacts in three patients) was available. For this study, a subset of 98 women (including 28 siblings) was selected for which both gene expression data and knee MR imaging data were available. The GARP study has been approved by the Medical Ethics Committee of the Leiden University Medical Center, the Netherlands (protocol nr. P76/98). All participants provided written informed consent to participate in the study.\n\nIn both RS and GARP, radiographs were scored to examine knee OA. Knee OA was defined as at least one definite osteophyte and definite joint space narrowing or at least two definite osteophytes (Kellgren and Lawrence (K/L) score ≥ 2).\n\nIn RS, all participants were scanned on a 1.5 T MRI scanner (General Electric Healthcare, Milwaukee, Wisconsin, USA) with an 8-channel cardiac coil, so that two knees could be scanned at once without repositioning the subject. The protocol consisted of a sagittal fast spin echo (FSE) proton density and T2 weighted sequence (repetition time (TR) = 4,900 ms; echo time (TE) = 11/90 ms, flip angle of 90–180, slice thickness 3.2 mm, field of view 15 cm2), a sagittal FSE T2 weighted sequence with frequency selective fat suppression (TR/TE = 6800/80 ms, flip angle = 90–180, slice thickness = 3.2 mm, field of view = 15 cm2), a sagittal spoiled gradient echo sequence with fat suppression (TR/TE = 20.9/2.3 ms, flip angle = 35, slice thickness = 3.2 (1.6) mm, field of view = 15 cm2) and a fast-imaging employing steady-state acquisition (FIESTA) sequence (TR/TE = 5.7/1.7 ms, flip angle = 35, slice thickness = 1.6 mm, field of view = 15 cm2). This FIESTA sequence was acquired in the sagittal plane. Total scanning time was 27 minutes for two knees per patient.\n\nAcquisition of MR imaging in GARP was performed using a 1.5 - T MR imaging scanner (Philips Medical Systems, Best, the Netherlands) using a 4-channel transmit/receive knee coil as described elsewhere13. The following images were obtained: coronal proton density- and T2-weighted dual spin echo (SE) images (with TR = 2,200 ms; TE = 20/80 ms; 5 mm slice thickness; 0.5 mm intersection gap; 16 cm field of view; 206 × 256 acquisition matrix); sagittal proton density- and T2-weighted dual SE images (TR = 2,200 ms; TE = 20/80 ms; 4 mm slice thickness; 0.4 mm intersection gap; 16 cm field of view; 205 × 256 acquisition matrix); sagittal three-dimensional (3D) T1-weighted spoiled gradient echo (GE) frequency selective fat-suppressed images (TR =46 ms; TE =2,5 ms; flip angle 40°; 3.0 mm slice thickness; slice overlap 1.5 mm; no gap; 18 cm field of view; 205 × 256 acquisition matrix); and axial proton density- and T2-weighted turbo spin echo (TSE) fat-suppressed images (TR = 2,500 ms; TE = 7.1/40 ms; echo train length 6,2 mm slice thickness; no gap; 18 cm field of view; 205 × 256 acquisition matrix). Total acquisition time (including the initial survey sequence) was 30 min for one knee per patient. Since the original purpose of the MR imaging study in GARP was to assess progression of OA, only one knee was imaged and no images were obtained of a knee that already had a maximum K/L score of 42.\n\nIn RS, a trained reader (who was blinded for any clinical, radiographic and genetic data) scored all MR images of the knees with the semi-quantitative Knee Osteoarthritis Scoring System (KOSS), described in detail elsewhere2. The joint effusion grades in the tibiofemoral joint (TFJ) and the patellofemoral joint (PFJ) were scored together (grade 0–3): 0 = joint effusion absent, 1 = small joint effusion, 2 = moderate joint effusion, and 3 = massive joint effusion. The scores of the left and the right knee were summed, resulting in one grade per person ranging from 0 to 6. An experienced musculoskeletal radiologist, also blinded for any clinical, radiographic and genetic data, scored a random sample of MR images to determine the inter-observer reliability. The inter-observer reliability was moderate to good with an intra-class correlation coefficient (ICC) of 0.83.\n\nIn GARP, MR images were also scored according to KOSS2 by three readers with 3, 15, and 25 years of experience in consensus, blinded to clinical, radiographic and genetic data, as described previously13. Presence of joint effusion was evaluated on T2-weigthed coronal, sagittal and axial sequences. A small, physiological sliver of synovial fluid was not recorded. A small effusion (grade 1) was present when a small amount of fluid distended one or two of the joint recesses, moderate effusion (grade 2) when more than two recesses were partially distended, and massive (grade 3) when there was full distension of all the joint recesses. As in RS, the grades were scored semi-quantitatively ranging from 0 to 3.\n\nBecause we used non-contrast-enhanced MR imaging in both GARP and RS, we could not measure synovial thickness reliably.\n\nIn RS, whole-blood was collected (PAXGene Tubes – Becton Dickinson) and total RNA was isolated (PAXGene Blood RNA kits - Qiagen). To ensure a constant high quality of the RNA preparations, all RNA samples were analyzed using the Labchip GX (Calliper) according to the manufacturer’s instructions. Samples with an RNA Quality Score > 7 were amplified and labelled (Ambion TotalPrep RNA), and hybridized to the Illumina HumanHT12v4 Expression Beadchips. Processing of the Rotterdam Study RNA samples was performed at the Genetic Laboratory of Internal Medicine, Erasmus University Medical Center Rotterdam, and the dataset has been deposited in the GEO database under the accession number GSE3382814.\n\nFor GARP, generation of gene expression levels in peripheral blood mononuclear cells (PBMCs) has been described elsewhere10. Gene expression data has been deposited in the GEO database under the accession number GSE48556.\n\nBoth RS and GARP samples were scanned on the Illumina iScan System (combined with an AutoLoader) using Illumina iScan image data acquisition software (version 3). Illumina GenomeStudio software (version 1.9.0) was used to generate output files for further statistical analyses. To identify transcripts that had detectable quantitative expression, we used the detection p-values reported by Illumina’s GenomeStudio software. The detection p-value represents the confidence that a given transcript is expressed above the background defined by negative control probes. We called a transcript significantly expressed when the detection p-value was <0.05 in more than 50 percent of all samples. All other transcripts were excluded from analysis. Because of this stringent detection p-value cut-off, the overall false-positive rate is very small (we won’t get false positive genes), whereas the false-negative rate might be higher (so we could lose some joint effusion associated genes, i.e., genes that are expressed at high joint effusion grades specifically).\n\nStatistical analyses were performed in R (version 3.1.2)15. Raw gene expression intensities were normalized by quantile-normalization to the median distribution; gene expression levels were subsequently log2-transformed. To minimize the influence of the overall signal levels, which may reflect RNA quantity and quality rather than a true biological difference between individuals, the probe means and sample means were centered to zero, and sample variance was linearly scaled, such that each sample had a standard deviation of one (standardization). To identify transcripts that were differentially expressed with joint effusion grades, we used four different linear regression models (lm):\n\n- Model 0: unadjusted: lm (probe ~ joint effusion grade)\n\n- Model 1: adjusted for age + fasting status + RNA quality score (RQS) + batch + cell counts\n\n- Model 2: adjusted for Model 1 + body mass index (BMI)\n\n- Model 3: adjusted for Model 1 + BMI + nonsteroidal anti-inflammatory drug (NSAID) intake\n\nBMI was measured at the research centers (as weight in kg divided by height2 in meters), and NSAID intake was extracted from the pharmacy records (RS) or collected via questionnaires (GARP). Because it is known that BMI is associated with markers of inflammation16,17, and because additional adjustments for NSAID use (model 3) hardly changed the effect sizes and standard errors of the results as shown in the Supplementary Table 1–Supplementary Table 2, we used model 2 for the meta-analysis and follow-up analyses. Notably, the analysis in GARP was also adjusted for siblingship in addition to age, batch, and BMI. In GARP, no adjustments were included for fasting status since blood was collected for all participants without fasting. Furthermore, gene expression levels were assessed from PBMCs and the RNA integrity number (or RQS) was at least 8.3 (36 random samples were analyzed)10.\n\nTo be able to meta-analyze the results of both studies, we combined the 12,843 Illumina HT12v4 probes (RS) and the 12,246 Illumina HT12v3 probes (GARP) based on chromosomal position and nucleotide sequence: 9,507 probes (representing 7,408 unique genes) were similar between the two gene expression platforms and could be meta-analyzed.\n\nWe ran sample size weighted meta-analyses based on p-values and the direction of the effects. By using the p-values and the effect direction, a Z-statistic characterizing the evidence for association was calculated. The Z-statistic summarized the magnitude and the direction of the effect. An overall Z-statistic and p-value was calculated from the weighted sum of the individual statistics. Weights were proportional to the square-root of the number of individuals examined in each sample and standardized such that the squared weights sum to 1. We used the Meta-Analysis Tool (version: generic-metal-2011-03-25) for genome-wide association scans (METAL)18 for this. METAL has been developed for meta-analyzing genetic genome-wide association studies. Because we are dealing with gene expression levels and not SNPs, we changed the SNPID column to probe IDs and assigned all probes a minor allele A and a major allele G, a minor allele frequency = 0.10, and a + strand. For the positions, the probe chromosomes and the midpoint position of the probes were used. Sample sizes, effect directions, and p-values were extracted from the linear regression model results files. Probes with a meta-analysis p-value<6.75E-06 (0.05/7,408 genes tested) were considered transcriptome-wide significantly associated with the joint effusion grades in the knee.\n\nPathway analysis was done with the DAVID tool; the Database for annotation, visualization and integrated discovery (version 6)19. We included all nominal significant genes (meta-analysis p-value <0.05), and checked for enrichment of any biological processes identified in the gene ontology database.\n\nTo investigate protein interactions among the nominal significant genes, we used the Search Tool for the Retrieval of Interacting Genes/Proteins (version 9.1)20, which is available online. With the “enrichment” option, we checked for enrichment of protein-protein interactions and “GO biological processes”.\n\n\nResults\n\nThe complete characteristics of the included subjects of both RS and GARP are shown in Table 1 and Figure 1. In both, RS and GARP, mean age of the subjects with and without joint effusion was not significantly different (ANOVA p-value RS = 0.146, ANOVA p-value GARP=0.181). Mean BMI seemed to be higher with higher joint effusion grades, but due to small sample sizes this difference was not significant (ANOVA p-value RS = 0.069, ANOVA p-value GARP=0.487).\n\n*this can be in one or two knees.\n\nJoint effusion grades in subjects without knee OA (A) and samples with knee OA (B).\n\nOf the 7,408 genes tested, CLEC4A (C-type lectin domain family 4, member A) demonstrated the strongest association with joint effusion grades in the knee (effect size=0.407 (SE=0.120); p-value =9.57E-04). In total, 310 probes (representing 251 unique genes) were nominally significant. The top 50 results are shown in Supplementary Table 1.\n\nIn GARP, the lowest p-value was found for the DNA-damage-inducible transcript 4 (DDIT4) gene (effect size=-1.425 (SE=0.411); p-value=5.21E-04). In total, 439 probes (representing 331 unique genes) were nominally significant (Supplementary Table 2).\n\nIn general, the top five genes of GARP and RS were different. To identify a common transcriptional signature for joint effusion, we performed a meta-analysis across RS and GARP. The top 20 results are shown in Table 2. All 257 nominally significant probes (representing 189 unique genes) are shown in Supplementary Table 3. The lowest p-value was found for the C1orf38 (Chromosome 1 Open Reading Frame 38) gene, also called THEMIS2 (Thymocyte Selection Associated Family Member 2) or ICB-1 (Induced by Contact to Basement membrane) (Zscore=-3.356; p-value=7.90E-04). Gene expression levels of C1orf38 were lower in samples with higher joint effusion grades in both whole blood and PBMCs (Supplementary Figure 1). Also the DYNLL2 gene (Dynein, Light Chain, LC8-Type 2), the NFATC1 gene (Nuclear factor of activated T-cells, cytoplasmic 1), and the RBM4 gene (RNA Binding Motif Protein 4) were nominally associated, with respectively higher (DYNLL2 and NFATC1) and lower (RBM4) gene expression levels correlating with advanced joint effusion grades (Supplementary Figure 2–Supplementary Figure 4).\n\nOf the 189 unique genes represented by the 257 nominally associated probes (p-value<0.05), 178 genes were recognized by the webtool DAVID. The most significant GO terms identified were: intracellular protein transport (GO:0006886: 13 of 374 genes, p-value=4.5E-04, Fold Enrichment (FE)=3.4), response to stress (GO:0006950: 34 of 1685 genes, p-value=1.5E-04, FE=2.0), antigen processing and presentation of exogenous antigens (GO:0019884: 4 of 14 genes, p-value=3.5E-04, FE=27.8), but the three GO terms did not survive the Benjamini Hochberg multiple testing correction. Additionally, one KEGG pathway was nominally significantly enriched: antigen processing and presentation (hsa04612: 5 of 83 genes, p-value=0.0127, FE=5.4).\n\nUsing the webtool STRING, we did not find significantly enriched protein-protein interactions within the network of 178 genes (p-value=0.386, observed interactions=58, expected interactions=55). However, STRING confirmed two significantly enriched biological pathways identified with DAVID: response to stress (45 of 1685 genes, p-value=6.23E-03) and antigen processing and presentation of exogenous antigens (10 of 14 genes, p-value=3.44E-02). The protein-protein interactions are visualized in Figure 2. Proteins involved in the antigen processing and presentation of exogenous antigens pathway (GO:0019884) are marked red, highlighting a cluster of three proteasomes (PSMA3, PSMD6, and PSME1) important for the antigen processing pathway.\n\nDisconnected proteins are hidden.\n\n\nDiscussion\n\nWe examined whether joint effusion grades in the knee were associated with specific gene expression levels in the circulation, which could potentially serve as molecular biomarker to indicate OA in the early stage. We identified 257 nominally associated probes (p-value<0.05) mapping to 189 unique genes. C1orf38, DYNLL2, and RBM4 were among the 5 most significant genes in the meta-analysis. Additional adjustments for BMI and NSAID intake did not notably affect the results, suggesting that the associations are consistent across all BMI ranges and in both users and non-users of NSAIDs. Subsequent pathway analyses with DAVID revealed nominal significant enrichment of genes involved in response to stress, gene expression, negative regulation of intracellular signal transduction, and antigen processing and presentation of exogenous antigens pathways. The biological pathways response to stress and antigen processing and presentation of exogenous antigens were confirmed with a second pathway analysis tool STRING.\n\nC1orf38 is a protein-coding gene and is highly expressed in several blood cells (monocytes, dendritic cells, NK-cells, T-cells, B-cells). The gene is induced by interferon-gamma (IFN-γ), an important cytokine that orchestrates many distinct cellular processes regarding inflammation21. Therefore, C1orf38 could be an interesting candidate for further research.\n\nCytoplasmic dynein consists of a molecular complex of several proteins including DYNLL2, and it is thought to play a role in movement and positioning of a wide range of organelles and complexes in the cell22. Notably, recent studies showed that DYNLL2 inhibits inflammation and may also inhibit osteoclastogenesis and bone resorption via regulation of NFκB transcription activity23. This would suggest that the higher expression of DYNLL2 in association with higher joint effusion grades is rather consequence than cause, however, this remains to be established.\n\nRBM4 is thought to play a role in alternative splice site selection during pre-mRNA processing, and seems to be important for the regulation of the translation of pro-inflammatory genes24.\n\nOf note is the association of higher joint effusion grades with higher expression levels of NFATC1 (nuclear factor of activated T cells 1). Besides its function in bone remodeling through calcium/calcineurin signaling, NFATC1 belongs to a family of transcription factors that play a central role in inducible gene transcription during immune response25. Although no significant differences were found in NFATC1 gene expression between OA-affected and unaffected tissues using microarray analyses26–29, a slight but significant reduction was detected by RT-qPCR in OA affected cartilage30. In addition, Jeffries and colleagues31 found changes in DNA methylation profiles, and it was shown that cartilage-specific ablation of NFATC1 predisposes to development of early onset OA too30. Since the expression of NFATC1 is positively associated with joint effusion it could be speculated that, in line with the increased expression of DYNLL2, upon occurrence of joint effusion specific pathways are activated to protect against development of OA. Consistent with this hypothesis, we observed that increased expression of NFATC1 in association with joint effusion is much more pronounced in subjects without knee OA in GARP. Therefore, NFATC1 might be a useful biomarker for early detection of OA. However, this should be confirmed in a longitudinal study tracking the development of the disease.\n\nThe pathway enrichment analysis results were consistent with known inflammatory disease mechanisms including response to stress and gene expression. Cellular stress and inflammation are known to reciprocally activate or inhibit each other, depending on the immune cell type and the stress-inducing signals32. Additionally, we identified the pathways negative regulation of intracellular signal transduction (GO:1902532) and antigen processing and presentation of exogenous antigens (GO:0019884). Hanada et al.33 already highlighted a key role for the intracellular signal transduction pathways of the pro- and anti-inflammatory cytokines which activate inflammatory transcription factors such as NF-κB, Smad, and STATs. The antigen processing machinery can be easily linked to the inflammatory response too34.\n\nSTRING showed the interaction between 3 proteasomes identified in the analysis (PSMA3, PSMD6, and PSME1). Proteasomes are important for degrading intracellular proteins, and recently it has been shown that mutations and polymorphisms in the proteasome are associated with several inflammatory and auto-inflammatory diseases35. Therefore, these genes could also be interesting targets for future studies.\n\nDespite the identification of several compelling potential markers for early OA, a major drawback of the current study was the relatively small sample size (n=233). Although gene expression data and knee MR images are available in larger datasets, the number of samples in which both measurements were determined is unfortunately limited. In addition, the data of the two cohorts (RS and GARP) was rather heterogeneous in particular due to the fact that in RS joint effusion grades were combined for two knees (sum of left and right knee), while in GARP joint effusion was determined in one randomly selected knee. Moreover, GARP is a cohort of clinical OA cases while RS is a population-based cohort study, in which no selection was made for OA cases specifically: in RS only seven out of 135 subjects (5.2%) were diagnosed with radiographically evident knee osteoarthritis, while in GARP 55 out of 98 subjects (56.1%) had knee OA. Furthermore, in RS the analyses were adjusted for fasting status (134 of 135 subjects fasted overnight) and RNA quality scores, while in GARP non-fasting subjects were used and RNA quality scores were available in a small subset only. Finally, gene expression levels in RS were determined in whole blood, while in GARP PBMCs were used. Although a previous study showed that expression levels differ across different RNA sources (whole blood, PBMCs, and lymphoblastoid cell lines), phenotype-based differential expression analyses results were consistent in whole blood and PBMCs36. Taken together, it is likely that cohort heterogeneity has resulted in limited power due to which no transcriptome-wide significant probes were identified. Earlier studies confirmed the good quality and reproducibility of our gene expression arrays10,27,37. Another potential limitation of our study is that we did not assess recent traumatic knee injuries: traumas can increase joint effusion and dilute our associations.\n\nIn conclusion, joint effusion grades in the knee on MR imaging were nominally associated with the expression levels of 189 unique genes in blood and the identified genes were mainly involved in inflammation. Although the associations presented in this manuscript were not transcriptome-wide significant, the meta-analysis and subsequent enriched biological pathways resulted in compelling candidate genes such as C1orf38 and NFATC1 that could be further characterized in future research. Additional studies are needed to replicate our findings as well as to identify other genes which will greatly help in understanding the pathophysiology of OA and its relation with inflammation, and may result in biomarkers urgently needed to diagnose OA at an early stage.", "appendix": "Author contributions\n\n\n\nAll authors have made substantial contributions to the completion of this study. Study concept and design: MP, YR, JM, IM. Acquisition of material and data: MP, YR, DS, AH, AU, EO, ES, MK, JB, SB, IM, JM. Data analysis: MP, YR, WH. Preparation of the manuscript: MP, YR, JM, IM. Critical reviewing and approval of the manuscript: all authors.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study was funded by the European Commission (HEALTH-F2-2008-201865, GEFOS; HEALTH-F2-2008 35627, TREAT-OA), the Netherlands Organization for Scientific Research (NWO) Investments (nr. 175.010.2005.011, 911-03-012), the Netherlands Consortium for Healthy Aging (NCHA), the Netherlands Genomics Initiative (NGI) / Netherlands Organization for Scientific Research (NWO) project nr. 050-060-810 and VIDI grant 917103521.\n\nThe Rotterdam Study is funded by Erasmus Medical Center and Erasmus University, Rotterdam, Netherlands Organization for the Health Research and Development (ZonMw), the Netherlands Organisation of Scientific Research NWO Investments (nr. 175.010.2005.011, 911-03-012), the Research Institute for Diseases in the Elderly (014-93-015; RIDE2), the Ministry of Education, Culture and Science, the Ministry for Health, Welfare and Sports, the European Commission (DG XII), and the Municipality of Rotterdam.\n\nThe generation and management of RNA-expression array data for the Rotterdam Study was executed and funded by the Human Genotyping Facility of the Genetic Laboratory of the Department of Internal Medicine, Erasmus MC, the Netherlands.\n\nThe GARP study was supported by the Leiden University Medical Centre and the Dutch Arthritis Association. Pfizer Inc., Groton, CT, USA supported the inclusion of the GARP study. The research leading to these results has received funding from the European Union's Seventh Framework Programme (FP7/2007-2011) under grant agreement n° 259679 and from BBMRI-NL, a research infrastructure financed by the Dutch government: NWO 184.021.007. Expression in blood was part of the Dutch Arthritis Association grant 10-1-402.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe are very grateful to Dr. E. Odding, Dr. A.P. Berging, Dr. M. Reijman, Dr. S. Dahaghin, Dr. H.J.M. Kerkhof, Ms. A. van Vaalen and Mr. M. Kool for scoring the knee radiographs.\n\nWe thank Ms. Mila Jhamai, Ms. Jeannette M. Vergeer-Drop, Ms. Bernadette van Ast-Copier, Mr. Marijn Verkerk and Jeroen van Rooij, BSc for their help in creating the RNA array expression database.\n\nWe are indebted to Dr. H.M. Kroon and Dr. N. Riyazi for scoring the radiographs and to Dr. P. Kornaat and Dr. R. Ceulemans for scoring the knee MR images.\n\nThe authors are very grateful to the study participants, the staff, the general practitioners, and the pharmacist from both participating studies.\n\n\nSupplementary Material\n\nSupplementary figures and tables for ‘Associations between joint effusion in the knee and gene expression levels in the circulation: a meta-analysis’.\n\nClick here to access the data.\n\n\nReferences\n\nDieppe PA, Lohmander LS: Pathogenesis and management of pain in osteoarthritis. Lancet. 2005; 365(9463): 965–73. PubMed Abstract \| Publisher Full Text\n\nKornaat PR, Ceulemans RY, Kroon HM, et al.: MRI assessment of knee osteoarthritis: Knee Osteoarthritis Scoring System (KOSS)--inter-observer and intra-observer reproducibility of a compartment-based scoring system. Skeletal Radiol. 2005; 34(2): 95–102. PubMed Abstract \| Publisher Full Text\n\nMathison DJ, Teach SJ: Approach to knee effusions. Pediatr Emerg Care. 2009; 25(11): 773–86; quiz 787–8. PubMed Abstract \| Publisher Full Text\n\nLo GH, McAlindon TE, Niu J, et al.: Bone marrow lesions and joint effusion are strongly and independently associated with weight-bearing pain in knee osteoarthritis: data from the osteoarthritis initiative. Osteoarthritis Cartilage. 2009; 17(12): 1562–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJohnson MW: Acute knee effusions: a systematic approach to diagnosis. Am Fam Physician. 2000; 61(8): 2391–400. PubMed Abstract\n\nAtukorala I, Kwoh CK, Guermazi A, et al.: Synovitis in knee osteoarthritis: a precursor of disease? Ann Rheum Dis. 2014; pii: annrheumdis-2014-205894. PubMed Abstract \| Publisher Full Text\n\nMabey T, Honsawek S: Cytokines as biochemical markers for knee osteoarthritis. World J Orthop. 2015; 6(1): 95–105. 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PubMed Abstract \| Publisher Full Text\n\nRiyazi N, Meulenbelt I, Kroon HM, et al.: Evidence for familial aggregation of hand, hip, and spine but not knee osteoarthritis in siblings with multiple joint involvement: the GARP study. Ann Rheum Dis. 2005; 64(3): 438–43. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKornaat PR, Bloem JL, Ceulemans RY, et al.: Osteoarthritis of the knee: association between clinical features and MR imaging findings. Radiology. 2006; 239(3): 811–7. PubMed Abstract \| Publisher Full Text\n\nWestra HJ, Peters MJ, Esko T, et al.: Systematic identification of trans eQTLs as putative drivers of known disease associations. Nat Genet. 2013; 45(10): 1238–43. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nR-Development-Core-Team. R: A language and environment for statistical computing. R Foundation for Statistical Computing. 2008. 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PubMed Abstract \| Publisher Full Text\n\nFranceschini A, Szklarczyk D, Frankild S, et al.: STRING v9.1: protein-protein interaction networks, with increased coverage and integration. Nucleic Acids Res. 2013; 41(Database issue): D808–15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTreeck O, Kindzorra I, Pauser K, et al.: Expression of icb-1 gene is interferon-gamma inducible in breast and ovarian cancer cell lines and affects the IFN gamma-response of SK-OV-3 ovarian cancer cells. Cytokine. 2005; 32(3–4): 137–42. PubMed Abstract \| Publisher Full Text\n\nPfister KK: Distinct functional roles of cytoplasmic dynein defined by the intermediate chain isoforms. Exp Cell Res. 2015; 334(1): 54–60. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim H, Hyeon S, Kim H, et al.: Dynein light chain LC8 inhibits osteoclast differentiation and prevents bone loss in mice. J Immunol. 2013; 190(3): 1312–8. PubMed Abstract \| Publisher Full Text\n\nBrudecki L, Ferguson DA, McCall CE, et al.: Mitogen-activated protein kinase phosphatase 1 disrupts proinflammatory protein synthesis in endotoxin-adapted monocytes. Clin Vaccine Immunol. 2013; 20(9): 1396–404. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTakayanagi H: Inflammatory bone destruction and osteoimmunology. J Periodontal Res. 2005; 40(4): 287–93. PubMed Abstract \| Publisher Full Text\n\nXu Y, Barter MJ, Swan DC, et al.: Identification of the pathogenic pathways in osteoarthritic hip cartilage: commonality and discord between hip and knee OA. Osteoarthritis Cartilage. 2012; 20(9): 1029–38. PubMed Abstract \| Publisher Full Text\n\nRamos YF, den Hollander W, Bovée JV, et al.: Genes involved in the osteoarthritis process identified through genome wide expression analysis in articular cartilage; the RAAK study. PLoS One. 2014; 9(7): e103056. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChou CH, Wu CC, Song IW, et al.: Genome-wide expression profiles of subchondral bone in osteoarthritis. Arthritis Res Ther. 2013; 15(6): R190. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLambert C, Dubuc JE, Montell E, et al.: Gene expression pattern of cells from inflamed and normal areas of osteoarthritis synovial membrane. Arthritis Rheumatol. 2014; 66(4): 960–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGreenblatt MB, Ritter SY, Wright J, et al.: NFATc1 and NFATc2 repress spontaneous osteoarthritis. Proc Natl Acad Sci U S A. 2013; 110(49): 19914–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJeffries MA, Donica M, Baker LW, et al.: Genome-wide DNA methylation study identifies significant epigenomic changes in osteoarthritic cartilage. Arthritis Rheumatol. 2014; 66(10): 2804–15. PubMed Abstract \| Publisher Full Text\n\nMuralidharan S, Mandrekar P: Cellular stress response and innate immune signaling: integrating pathways in host defense and inflammation. J Leukoc Biol. 2013; 94(6): 1167–84. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHanada T, Yoshimura A: Regulation of cytokine signaling and inflammation. Cytokine Growth Factor Rev. 2002; 13(4–5): 413–21. PubMed Abstract \| Publisher Full Text\n\nKasajima A, Sers C, Sasano H, et al.: Down-regulation of the antigen processing machinery is linked to a loss of inflammatory response in colorectal cancer. Hum Pathol. 2010; 41(12): 1758–69. PubMed Abstract \| Publisher Full Text\n\nGomes AV: Genetics of proteasome diseases. Scientifica (Cairo). 2013; 2013: 637629. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJoehanes R, Johnson AD, Barb JJ, et al.: Gene expression analysis of whole blood, peripheral blood mononuclear cells, and lymphoblastoid cell lines from the Framingham Heart Study. Physiol Genomics. 2012; 44(1): 59–75. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPeters MJ, Joehanes R, Pilling LC, et al.: The transcriptional landscape of age in human peripheral blood. Nat Commun. 2015; 6: 8570. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "13228", "date": "15 Apr 2016", "name": "Richard Barrett-Jolley", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 well conducted study that addresses a very important, and topical question. The discovery of a biomarker of osteoarthritis in the blood would be somewhat of a Holy Grail in the field and so it is important that investigators look for suitable correlations within datasets such as the Rotterdam Study. I have no major concerns with this study, but have a number of comments and also some small points that the authors may be able to clarify.A practical limitation of studies such as these, comparing biomarker level data from patients with higher levels of joint effusion already, is that OA may have already progressed to a point where a biomarker is no longer so useful. That said, on its discovery, such a biomarker would then of course be a good candidate to analyse at earlier stages of OA to then determine if it is a useful OA predictor prior to MRI-level changes. I was curious to know, if long term, these authors will be able to correlate expression levels of genes expressed in blood of healthy people which then end up with OA at a later stage? It has emerged over recent years that “osteoarthritis” probably represents a family of conditions, some of which have a strong component of inflammation and some which do not. I was not sure that the distinction between inflammation being an important contributor to all OA or just some subtypes of OA was clearly made in the introduction. My understanding is that it is the latter. The authors comment that this is a small sample size at some point. I was not really sure it was. I realise that for some GWAS, 1000s of patients are necessary to detect small odds ratios; however, I thought that if a biomarker was to be useful diagnostically, the change would have to be substantial enough that it should manifest statistically significant differences, even in just a few patients. Therefore, I felt, in the context of this study, it was an unnecessary self-criticism. I applaud the authors for already getting the new data in the GEO database, some groups take many years to upload this, which can be frustrating. I did miss, initially that this meta-analysis pertained to expression datasets uploaded in May and July 2013. Somehow (I think it was reference to RNA extraction methods), at first, I thought this had been newly acquired. Would the words “pre-existing datasets” be useful in the methods, or would most people consider the word “met-analysis” in the title means all the data is pre-existing? The definition of “significant” looked a little arbitrary to me. I know p<=0.05 is arbitrary in itself, but it has been a standard for many years now. But 0.05 in > 50% of samples seemed strange to me. The authors state that the overall FDR is very small, but what is it? Can it be calculated or estimated? I think the “(we won’t get false positive genes)” could be deleted? Benjamini Hochberg correction is used later in the pathway analysis, but not here in the regression/differential gene detection section. Is there a good reason for this? In the differential expression section (not the regression) “Effect Size” is not being used in the normal context I use it. Does it mean “ratio”? Table 2; the key table in the study. I felt the legend could be more helpful, and include more detail? Table 2: “Top genes”? “top” in what way? Lowest p-val? Largest change? My feeling is that the pathway analysis basically revealed no significantly enriched pathways, once you excluded multiple comparison error? I thought that was quite interesting (and negative data is of course valuable), but if I am correct in my interpretation, this is most certainly not stated clearly in the abstract or elsewhere.", "responses": [] }, { "id": "13226", "date": "18 Apr 2016", "name": "Csaba Matta", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIdentifying and validating biomarkers from easily accessible body fluids such as peripheral blood for early (pre-radiographic) detection of osteoarthritis (OA), or at least OA susceptibility, are of key importance in the field. The authors of this manuscript aimed to analyse gene expression levels associated with joint effusion in two cohorts of patients either in the Genetics, Arthrosis and Progression study (GARP) or in the Rotterdam study (RS). This is a very well presented and conducted study, with results that may hopefully advance the field.In my opinion, the major limitation of this study (which is actually stated and recognised by the authors as well) is that the two cohorts on which the study was performed are very different in nature; GARP is a cohort of clinical OA cases, whilst RS is a population-based study. This might be the reason as to why the top 5 genes in the two cohorts were different. Perhaps it would be useful to check whether the top genes in the GARP study (in which more than 50% of the subjects had OA) have any predictive value for pre-radiographic OA in the RS study. This question could of course only be answered over time.Following on the comment of the first referee regarding the inflammatory/non-inflammatory OA phenotypes, it would also be interesting to see whether the same GO pathways would be identified if patients with inflammatory/non-inflammatory OA were separately analysed.", "responses": [] }, { "id": "13227", "date": "18 Apr 2016", "name": "Gauthaman Kalamegam", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 investigates the associations between joint effusion in the Knee and gene expression levels in the circulation. This is a well performed study that had led to the identification of 189 unique genes in blood with joint effusion in knee and few candidate biomarkers. Although the sample size was less and had cohort heterogeneity the authors have made a very systematic approach to identify genes and biological pathways involved in osteoarthritis.", "responses": [] } ]	1	https://f1000research.com/articles/5-109
https://f1000research.com/articles/5-108/v1	27 Jan 16	{ "type": "Review", "title": "Recent Understandings of Pet Allergies", "authors": [ "Dennis Ownby", "Christine Cole Johnson", "Christine Cole Johnson" ], "abstract": "Allergic reactions to pets have been recognized for at least a hundred years. Yet our understanding of the effects of all of the interactions between pet exposures and human immune responses continues to grow. Allergists, epidemiologists, and immunologists have spent years trying to better understand how exposures to pet allergens lead to allergic sensitization (the production of allergen-specific immunoglobulin class E [IgE] antibodies) and subsequent allergic disease. A major new development in this understanding is the recognition that pet exposures consist of not only allergen exposures but also changes in microbial exposures. Exposures to certain pet-associated microbes, especially in the neonatal period, appear to be able to dramatically alter how a child’s immune system develops and this in turn reduces the risk of allergic sensitization and disease. An exciting challenge in the next few years will be to see whether these changes can be developed into a realistic preventative strategy with the expectation of significantly reducing allergic disease, especially asthma.", "keywords": [ "Pet Allergies", "Allergic reactions", "IgE antibodies", "allergic sensitization" ], "content": "Introduction\n\nMany families in the US and other countries keep a variety of pets. The American Pet Products Association estimates that 65% of US households own a pet, an increase of 3% from 2010 to 2015. Although this estimate may be high, there is little doubt that approximately half of all American families have a pet and the vast majority of these pets are furry1,2. It is also well known that when there is a high prevalence of pet-keeping in a community, pet allergens are found in relatively high concentrations in public places such as schools. These “second-hand” exposures to pet allergens have been shown to exacerbate disease in sensitive children3. The purpose of this review is to provide an understanding of some of the recent studies related to pet allergies and potential health consequences. The areas to be explored include the following: the changing prevalence of all types of allergic diseases, how people’s interactions with pets have changed, new concepts of how the human immune system responds to pet allergens, and especially the growing understanding of how this immune response relates to the microbial ecology of the gut. The potential economic costs of pet allergies will also be briefly explored. There are many excellent reviews focusing on the precise mechanisms of human immune responses to pets4–9.\n\nThis review will focus on scientific research related to human allergic responses to pets. However, it is important to recognize that pet allergy can be a very emotionally sensitive topic. Every practicing allergist has repeatedly heard a family say that “if ‘X’ person in the family is allergic to the pet, then ‘X’ goes and the pet stays in the home!”. This statement is always meant to be humorous but it clearly comes from strong feelings about the pet’s place in the family. This is but one example of how pets are commonly considered members of the family and considerable angst is generated if there is a conflict between the potential health of a family member and the love of the pet. The point to be made is that strong emotions may lead to strongly held beliefs that have little factual basis. The internet has widely spread information on topics such as “low” or “hypoallergenic” pets when there is little supporting scientific evidence10,11.\n\nThis review will focus on cats and dogs since these are the most popular pets in the US and many other developed countries. Because cats and dogs are the most prevalent household pets, there are many more studies of how they relate to allergic disease. Although reactions to other animals will not be examined in detail, allergic responses to other animals are believed to be similar to those elicited by cats and dogs after considering the relative intensity and duration of exposures, and many studies have shown that the majority of pet allergens come from the same protein family12,13. An important cross-over area is occupational exposure versus in-home exposure to animals. For example, in both settings, exposure to mice or rats can be desired (pets or reared animals) or unwanted (vermin). The literature in these areas is voluminous but beyond the scope of this review.\n\nAn important topic that is beyond this review is the relationship between exposure to cats and dogs and the risk of allergic disease. This subject has been widely debated and subjected to numerous reviews but will not be included herein4,14–19.\n\n\nTemporal increase in prevalence of allergies\n\nMany studies over the previous two to three decades have suggested that allergic diseases have increased in frequency20. Most notable has been the increase in asthma over the past three decades. Although some would argue that there have been epidemics of different allergic diseases at different times, it is difficult to discern how many of these changes were related to gradually improving recognition and diagnosis of new diseases20. However, there is little doubt that asthma has nearly tripled in prevalence among youth in the US since the 1970s and that food allergies have at least doubled in the same peroid21,22. The reasons for the increase in the prevalence of allergies have been widely debated and investigated but there is no consensus on the precise cause. Among the many hypotheses are improving hygiene, global warming, increasing use of antibiotics (especially in food), and reduced physical activity. One of the few agreed-upon assumptions is that the increase has occurred during one or two generations, making genetic evolution highly improbable; however, a new area of inquiry is epigenetic change, which is the heritable change in gene expression that does not involve changes to the underlying DNA sequence. There are at least three mechanisms of epigenetic change: DNA methylation, histone modification, and non-coding RNA-associated gene silencing23. Epigenetic changes have been linked to asthma in some studies24–27.\n\n\nChanging relationships of people to pets\n\nObjective data concerning how persons view their pets and the role of pets in families over multiple generations are extremely hard to find. A study by Kennedy and McGarvey took an interesting approach to examining this question of whether the relationship between pets and their families has changed over time28. They examined 1,348 advertisements including both people and pets which had appeared in popular US women’s magazines from the 1920s through the 1980s and coded the ads for seven themes such as whether the pet was depicted indoors or outdoors, was on a leash, or was used to depict companionship (i.e. touching or holding the pet). They concluded that over the time interval studied, pets have moved from outdoor protectors to indoor family members28.\n\nThe increasingly close contact between persons and pets and the resulting higher allergen exposures occurred over the same decades during which all allergic diseases appeared to be increasing. This combination has been thought to be a major reason for the increase in pet allergies. Unfortunately, data supporting this hypothesis are not robust. In the US, skin test results from the NHANES (National Health and Nutrition Examination Survey) II and III studies were compared. NHANES II was conducted from 1976 through 1980, and NHANES III was from 1988 through 1994. There were six allergens common to both studies, allowing comparison of results in a representative sample of the non-institutionalized, US population, from ages 6 to 59 years. The probability of reacting to at least one allergen skin test was higher in NHANES III compared with II: 41.9% (standard error [SE] 1.23) and 21.8% (SE 0.94), respectively, slightly more than a doubling. Reactivity to cat, the only pet allergen tested in both NHANES II and III, increased 5.5-fold (3.1% to 17.0%) compared with an average increase of 2.6-fold for the other five in common allergens (rye and Bermuda grasses, short ragweed, oak, and Alternaria alternate). Tempering this finding is the possibility that the cat allergen used in NHANES III was more potent, thus increasing reactivity; however, studies from Europe suggest similar changes29.\n\n\nImmune response to pet allergens\n\nAllergy has always been defined by the presence of immunoglobulin class E (IgE) antibodies immunologically specific for individual antigens. Initially, the minute quantities of IgE present in humans could be detected only by allergen skin testing. Now laboratory tests for cat allergens are essentially as sensitive as skin tests, but the results of skin and in vitro tests are not always identical29,30. Antigens eliciting IgE responses are referred to as allergens. Although IgE antibodies have been most extensively studied, humans do produce other immune responses to allergens. Immune responses are initiated by specialized antigen-presenting cells such as dendritic cells, which present the allergen to T cells. Attempts have been made to identify the small portions of the major cat allergen (Felix domesticus 1, abbreviated Fel d 1) presented to T cells, in the hope of using these peptides to induce hyporesponsiveness to Fel d 1 as a treatment for cat allergy31,32. Recent studies have identified how the cysteine-rich portion of the major cat allergen, Fel d 1, is bound on cells through a mannose receptor33. Some have suggested that Fel d 1 is uniquely able to induce an IgG subclass 4 (IgG4) response in many individuals and that high concentrations of Fel d 1–specific IgG4 can block IgE responses34,35. However, other studies have not found a relationship between cat-specific IgE and IgG4 levels and symptoms36,37. Multiple studies, including those of allergen immunotherapy with cat and other allergens, all suggest that repeated relatively high-dose exposure to any allergen leads to IgG4 production7,38,39.\n\nInterestingly, the allergens characterized from furry animals thus far have all belonged to three broad groups of proteins: secretoglobins, lipocalins, and kallikreins. Whereas Fel d 1 is a secretoglobin of unknown function, more than 50% of allergens from furry animals have been identified as lipocalins12,13. These animal allergens are found in dander, saliva, and urine. They are commonly on small particles that allow airborne dispersion and also dispersion by adherence to surfaces such as clothing2,40. The apparent constant circulation of pet allergens on shoes and clothing through public areas and into homes has made it very difficult to control symptoms from pet allergens by avoidance measures such as air cleaning41,42.\n\n\nMicrobial exposures related to pets and other animals\n\nProbably the most dramatic change in understanding the relationships between pet exposure and pet allergy is the realization that pet exposure involves more than just exposure to the allergens shed by the pet43. Multiple studies have shown that early life exposure to pets and to farm animals is associated with a reduced risk of subsequent allergic disease44–46. Although other studies have disputed these findings, the results of systematic reviews and meta-analyses have typically shown either a reduction or no increase in risk associated with infantile exposure to furry pets47–50. The hypothesis developed to explain why animal exposure could be associated with a decreased prevalence of allergy postulates that animals increase the diversity of microbes to which a child is exposed, and that this more diverse exposure leads to the development of an immune system less likely to develop allergic responses to antigens. Two studies have demonstrated that cats or dogs in the home increase the diversity of the microbiota of the home43,51. Another study showed that the stool microbiota of children living with pets differed from those without pet exposure. Studies in homes of farmers also suggest a broader diversity of microbes52–54. Several investigations have been directed toward understanding the dominant exposures of farm living leading to a lower prevalence of allergic disease. One important factor in farm living is consumption of farm (i.e. unpasteurized) milk55–57. The assumption is that farm milk contains many live bacteria that can alter the gut microbiota of the child, or that unpasteurized milk contains substances supporting the growth of specific microbes. This hypothesis is supported by studies showing that the amount of bacterial contamination in surface water used for drinking is directly correlated with a lower risk of allergic disease58. Variables that appear relatively consistent in all of these studies are (1) that the exposure to diverse bacteria must occur in during the first year of life and perhaps in the first weeks of life, and (2) that the types of bacteria which appear to be protective are common soil bacteria or bacteria found in the gastrointestinal tracts of mammals. This increasing knowledge related to microbial exposure has led some to suggest that we shift from the “hygiene” to the “microbial” hypothesis of allergen protection59,60. The critical question is whether this knowledge can be developed into a medically valuable preventive strategy such as supplementing mothers or infants with live bacteria at a critical stage of development.\n\nAlthough the full demonstration of this animal-microbe-gut-immune development hypothesis has not been achieved, multiple human and animal studies strongly support the hypothesis. Several early studies have shown that there are differences in stool microbes between children in certain countries and that these changes are associated with the risk of asthma61. One of the most supportive mouse studies was by Fujimura et al.62. These investigators first compared gavaging young adult mice with slurries of house dust from homes with and without dogs. The mice were then immunized with cockroach allergen by using a protocol designed to induce allergic sensitivity and asthma-like airway reactivity. The mice given the dust from homes with a dog were strongly protected from sensitization and airway disease compared with mice given the house dust from homes without dogs. An analogous experiment using challenge with respiratory syncytial virus (RSV) again showed strong protection of the mice given dust from homes with dogs. When the microbial communities of the caeca of the gavaged mice were examined, several different microbes were in much higher abundance in the mice given dust from homes with dogs. One of these bacteria, Lactobacillus johnsonii, was cultured and given to groups of mice. The mice given gavages with live L. johnsonii were again significantly protected from both allergen-induced and RSV infection-induced airway disease, but gavages with killed bacteria were not effective. It has been shown that supplementing high-risk infants with Lactobacillus casei subsp. Rhamnosus (LGG) does alter the development of the gut microbiome and so it may be possible to use supplementation as a disease prevention strategy in humans63. Others have shown similar protective effects from Lactobacillus reuteri in mice64. There are also suggestions that vitamins and other diet elements may play roles in altering the gut microbiome and the subsequent function of the immune system65,66.\n\nWhile the hypothesis of infants acquiring a different gut microbiome from animal exposure has been developing, there are other findings that suggest this hypothesis is missing essential elements. An alternative hypothesis is that pregnant women living with pets or closely associated with farm animals may over time develop different gut and vaginal microbiomes and that these pet-associated maternal gut microbiomes are inoculated into infants during normal vaginal birth. This would fit with the studies showing that birth by C-section carries a higher risk of childhood asthma than vaginal birth, presumably because an infant does not acquire a large inoculum of maternal vaginal and gut microbes at birth67–69. Another study related to this hypothesis is the finding that the prenatal presence of dogs in homes has a stronger effect on the development of total serum IgE in infants delivered by C-section than those born vaginally70. It could be argued that if pets were associated with an alteration of the maternal gut or vaginal microbiome, a stronger effect would have been expected in infants born vaginally. An alternative argument is that infants born by C-section have a suboptimal initial maternal inoculum, which allows a greater impact by environmental microbes such as those in house dust on early colonization68,71,72.\n\nA common argument against the hygiene and microbial hypotheses related to allergy is the frequently quoted high prevalence of asthma among inner-city residents73–75. However, a recent study from the Inner-City Asthma Consortium found a clear interrelationship between allergen exposure, microbe exposure, and risk of disease18. In that study, children with the greatest exposure to allergens and bacteria in specifically the first year of life had the lowest risk of recurrent wheeze and allergic sensitization, again suggesting the protective effect from exposure to a high diversity of bacteria. Unfortunately, that study was racially homogenous and so potential effects of race could not be evaluated.\n\nAn important element to consider in all studies of pets and of microbes is the timing of exposure related to immune development. As already suggested, the effects of pets on the development of the infant gut microbiome are likely to be much larger in the first weeks and months of life than in later childhood. The one-year age cutoff found in many studies may be an artifact of how data were collected rather than a biological horizon.\n\nA common question related to the apparent protective effect of early pet exposure on allergy is whether this is of clinical significance and if so how the information should be used. The current level of understanding is inadequate at best. If our hypothesis that early pet exposure may alter an infant’s developing microbiota and lead to a reduced risk of some immune diseases is correct, then the critical question is whether this knowledge can be transformed into a therapeutic strategy. Clearly, exposing all children to pets is not possible and probably not desirable. There are many questions related to owning a pet to consider: costs of food, veterinary care, possible zoonotic infections, etc. These are questions that persons should carefully consider before obtaining a pet. However, if a pregnant woman has a pet when she finds that she is pregnant, we believe that there is no increased risk of allergic disease and probably a decreased risk if she continues to keep the pet through the birth of her child and the child’s first year. After the first year of life, the data become inconsistent. Some studies suggest that continued pet exposure after the first year of life provides additional protection from atopy whereas other studies do not find any benefit after the first year76–78. The more important question is whether knowledge of the interaction of pet and human microbiota can be used to provide a preventive option such as a probiotic supplement. Although such trials have been conducted with mixed results, it appears that much more study and understanding are necessary before there will be consistent success with such approaches79,80. Other potentially simple approaches to allergy reduction that may be related to microbial exposures, such as hand washing of dishes and licking pacifiers, seem to be helpful with minimal risks81,82.\n\n\nHealth-care costs related to pet allergy\n\nThe potential allergy-related health-care costs of keeping pets are rarely discussed in the medical literature, partially because these costs are difficult to objectively assess. One question is whether pet-keeping by pet-allergic individuals with asthma substantially increases costs of asthma care. A study estimated that the increased number of visits for acute asthma care among dog-allergic adults, who chose to live with dogs, might add as much as $0.5 to 1.0 billion per year to costs of care in 201083. This estimate suggests a substantial increase in health-care cost for adults but does not include indirect costs such a lost work days which would drive the estimate even higher83. Unfortunately, there have not been any similar estimates of how pet allergy might increase the cost of asthma care for children. However, Almqvist et al. showed that even indirect exposure to cat dander, brought into classrooms on the clothes of children living with cats, increased symptoms and medication use in cat-allergic children with asthma84. The increased costs of new-onset asthma in a child are also difficult to estimate but substantial. A longitudinal study of 3,535 school children in California identified only three risk factors for new-onset asthma in these children: a humidifier (relative risk [RR] 1.7, confidence interval [CI] 1.2–2.4), any pet (RR 1.6, CI 1.0–2.5), and having a dog in the home (RR 1.4, CI 1.0–2.0)85. Similarly, in other studies, sensitization to cats or dogs has been identified as a risk factor for new-onset reactive airway disease86,87. The presence of a dog in the home also increased ozone exposure-related asthma symptoms in a study88. In total, these studies suggest that allergic sensitivity to pets and pet exposure are significant contributors to the overall costs of asthma care.\n\nThe combination of the studies summarized in this review shows a somewhat paradoxical relationship of pets and allergy. Exposure to the microbes associated with pets in the first few months of life appears to be associated with a substantial reduction in the risk of allergic disease and asthma. This effect appears to last at least until early adulthood78. Only one longitudinal study has shown that continuing exposure to dogs was required for continuing protection at least until 7 years of age76. Others have not shown any apparent effect after the first year77. A few studies have shown that sensitization to cats or dogs is a risk factor for new-onset asthma later in life, and one study has shown that the presence of a dog in the home was a risk factor for new-onset asthma.\n\n\nSummary\n\nPets are an important source of health benefits to many individuals. But close contact with pets, such as when they live in homes, can be associated with a variety of risks, including medically significant allergic diseases. Adding confusion to our understanding of the relationship of pets to allergic disease has been the discovery that infantile exposure to furry pets appears to be associated with a substantial reduction of allergy and asthma risks in childhood; however, it is possible that continuing pet exposure may become a risk for allergies and asthma at some stage of life. The apparent allergy-protective effect of pets appears to be mediated through exposure to a more diverse microbial community in the home. The discovery of this microbe-related protective effect will hopefully lead to allergy prophylactic options in the coming years without requiring direct pet or other animal exposure.", "appendix": "Competing interests\n\n\n\nNeither of the authors has any commercial competing interests related to the ideas and opinions expressed in this manuscript.\n\n\nGrant information\n\nThe authors are funded by the National Institutes of Allergy and Infectious Disease for studies related to the relationships between the human gut microbiome and allergic disease.\n\n\nReferences\n\nZane JP: Pets of the golden years. New York Times. New York, 2015.\n\nArbes SJ Jr, Cohn RD, Yin M, et al.: Dog allergen (Can f 1) and cat allergen (Fel d 1) in US homes: results from the National Survey of Lead and Allergens in Housing. J Allergy Clin Immunol. 2004; 114(1): 111–7. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHesselmar B, Sjöberg F, Saalman R, et al.: Pacifier cleaning practices and risk of allergy development. Pediatrics. 2013; 131(6): e1829–37. PubMed Abstract \| Publisher Full Text\n\nHesselmar B, Hicke-Roberts A, Wennergren G: Allergy in children in hand versus machine dishwashing. Pediatrics. 2015; 135(3): e590–7. PubMed Abstract \| Publisher Full Text\n\nOwnby DR: Pet dander and difficult-to-control asthma: The burden of illness. Allergy Asthma Proc. 2010; 31(5): 381–4. PubMed Abstract \| Publisher Full Text\n\nAlmqvist C, Wickman M, Perfetti L, et al.: Worsening of asthma in children allergic to cats, after indirect exposure to cat at school. Am J Respir Crit Care Med. 2001; 163(3 Pt 1): 694–8. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nMcConnell R, Berhane K, Gilliland F, et al.: Indoor risk factors for asthma in a prospective study of adolescents. Epidemiology. 2002; 13(3): 288–95. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nLitonjua AA, Sparrow D, Weiss ST, et al.: Sensitization to cat allergen is associated with asthma in older men and predicts new-onset airway hyperresponsiveness. The Normative Aging Study. Am J Respir Crit Care Med. 1997; 156(1): 23–7. PubMed Abstract \| Publisher Full Text\n\nHedman L, Andersson M, Bjerg A, et al.: Environmental risk factors related to the incidence of wheeze and asthma in adolescence. Clin Exp Allergy. 2015; 45(1): 184–91. PubMed Abstract \| Publisher Full Text\n\nMcConnell R, Berhane K, Molitor J, et al.: Dog ownership enhances symptomatic responses to air pollution in children with asthma. Environ Health Perspect. 2006; 114(12): 1910–5. PubMed Abstract \| Free Full Text" }	[ { "id": "12106", "date": "27 Jan 2016", "name": "Wanda Phipatanakul", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12107", "date": "27 Jan 2016", "name": "David Lang", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-108
https://f1000research.com/articles/5-106/v1	26 Jan 16	{ "type": "Review", "title": "Sequence co-evolutionary information is a natural partner to minimally-frustrated models of biomolecular dynamics", "authors": [ "Jeffrey K Noel", "Faruck Morcos", "Jose N Onuchic", "Jeffrey K Noel", "Faruck Morcos" ], "abstract": "Experimentally derived structural constraints have been crucial to the implementation of computational models of biomolecular dynamics. For example, not only does crystallography provide essential starting points for molecular simulations but also high-resolution structures permit for parameterization of simplified models. Since the energy landscapes for proteins and other biomolecules have been shown to be minimally frustrated and therefore funneled, these structure-based models have played a major role in understanding the mechanisms governing folding and many functions of these systems. Structural information, however, may be limited in many interesting cases. Recently, the statistical analysis of residue co-evolution in families of protein sequences has provided a complementary method of discovering residue-residue contact interactions involved in functional configurations. These functional configurations are often transient and difficult to capture experimentally. Thus, co-evolutionary information can be merged with that available for experimentally characterized low free-energy structures, in order to more fully capture the true underlying biomolecular energy landscape.", "keywords": [ "minimally frustrated models", "biomolecular dynamics", "frustrated protein models", "protein structure model", "x-ray crystallography", "nuclear magnetic resonance", "Direct coupling analysis" ], "content": "Introduction\n\nHigh-resolution structural techniques (e.g., X-ray crystallography and nuclear magnetic resonance) have provided the data necessary to develop and refine a multitude of potential energy functions used in the simulation of biomolecules. In particular, these structures provide the parameterization for simplified models that are based on the energy landscape theory of protein folding. These models construct an energetically unfrustrated (ideal) funneled landscape by including stabilizing interactions between native contacts (i.e., amino acid pairs that are nearby in the three-dimensional native structure of a protein). In cases in which experimental structures are lacking or insufficient, it becomes necessary to supplement these models with other sources of contact information. An emerging technique for contact estimation is via the statistical analysis of residue co-evolution in families of protein sequences. Combinations of high-resolution structural data and predictions from residue co-evolution are proving to be invaluable tools for building models to study protein structure and dynamics.\n\nUnderstanding the fundamental process of how a heterogeneous polypeptide can reversibly fold into a distinct native three-dimensional structure on biological timescales led to the development of the energy landscape theory of biomolecular folding. This theory is based on the principle of minimal frustration1 and the folding funnel concept2,3. These physical principles describe an energy landscape that has been molded by evolution such that the native interactions (i.e., the molecular interactions present in low free-energy configurations of folded proteins and RNAs) are, on average, more stabilizing than non-native interactions. The consequence of proteins having sufficiently reduced energetic frustration is that geometry dominates energetic roughness in determining folding mechanisms. Thus, a description of the effective energetics of the folding phenomenon can be attained by including a set of native stabilizing interactions consistent with the native basin of attraction. Potential energy functions of this type, which use experimental information to determine such native interactions, are known as “structure-based models” (SBMs)4–6 and, when employed in dynamical models, are powerful tools for understanding the connection between structure, folding, and function. Although these SBMs have been successfully applied to different biomolecules, we will be focusing on proteins for clarity in this review.\n\nStructural information, however, may be limited for many interesting systems. This is particularly true for functional configurations that are transient or partially disordered or both. The recent explosion in genomic information has enabled complementary methods for discovering functionally important amino acid interactions. The minimal frustration principle applies equally to any sequence of amino acids that can robustly fold to a particular native structure. Thus, in a family of sequences where most of them fold to a common structure, residue positions that are in contact will display a correlated mutational record because of the global evolutionary constraint that the native structure imposes for foldability. Of course, additional constraints beyond folding affect sequence evolution, including maintenance of molecular assemblies, enzymatic activity, and allosteric motions. Signals of these functionally relevant contacts are necessarily mixed with those providing robust folding. To identify such relevant interactions involved in folding and function, a number of methodologies have been developed in recent years that have been successful in uncovering such molecular couplings from sequence data. One of them is direct coupling analysis (DCA)7,8, which is designed to infer a global statistical model from a multiple sequence alignment (MSA) of a single protein family. Using a maximum entropy approach, DCA infers the parameters of an effective energy function consisting of single-site fields and pairwise couplings that is able to approximately reproduce the empirically observed single-site and pairwise amino acid frequencies from the input sequence alignment. The DCA energy function is known as a Potts model, a generalized Ising model that includes non-nearest neighbor interactions and non-constant spin-spin interactions. In practice, couplings of varying strength are computed between all possible pairs of sequence positions. In the past, accurate and tractable approximations of such global models were elusive and detection of direct correlations, as opposed to an aggregate of direct and indirect correlations, was challenging. Other methods are derived from similar theoretical perspectives but have varying computational demands and accuracies9–12. Using an inferred effective energy function, one can estimate pairwise direct probabilities at a particular pair of residue sites. Calculating the Kullback–Leibler divergence between these joint probabilities and single marginal frequencies gives the direct information (DI) score for that residue pair. DI is a proxy of how “directly correlated” two sites are in an MSA. When compared with crystal structures, high DI scores correlate highly with native contacts, and more than 80% overlap, on average, for the top residue pairs in many protein families7,13. The full set of highly scoring contacts amounts to a superset of minimally frustrated and functionally important residue pairs that are spatially localized in the functional configurations of the members of a protein family. Here, we will review the current progress in using residue co-evolution for modeling the structure and dynamics of proteins with a focus on its combination with SBMs.\n\n\nResidue co-evolutionary constraints are natural input for minimally frustrated protein models\n\nIn their simplest form, SBMs idealize minimally frustrated protein energy landscapes by including only native interactions. This model removes any residual non-native energetic roughness and clarifies analysis of the geometrical and topological aspects of protein dynamics and folding. These models faithfully represent the local geometry through bond, angle, dihedral, and excluded volume terms at either single-bead-per-residue or all-atom resolutions. Non-local interactions consist of stabilizing pairwise potentials applied between residue (or atom) pairs that are nearby in the native structure. These pairwise interactions are called native contacts, and the entire set is known as a native contact map. All of the interactions, local and non-local, are set to have an explicit minimum at the native structure, hence the name “structure-based”. The simplified construction of the potential energy function permits for reduced computational requirements, and the explicitly encoded native interactions provide a baseline model that can be used for molecular modeling or studying physical perturbations. For a detailed discussion of the theoretical foundation and construction of SBMs, we refer you to the following reviews,14–16, and the references therein.\n\nThe quality of contact maps derived from DCA and similar methods have been benchmarked against contact maps calculated from crystallographic structures, and their accuracy is promising. In general, the larger and more diverse the family of sequences, the higher the quality of contact prediction. The high level of DCA accuracy provided sufficient tertiary constraints to allow folding single domain proteins to within 3 Å from the crystal structure when given knowledge of the secondary structure17–21. A rule of thumb is that the number of sequences should be larger than 1000 with less than 80% identity; however, others propose an even lower requirement of a minimum number of sequences close to the length L of the protein polypeptide chain, provided that they are diverse18. The notoriously difficult problem of predicting membrane protein structures has also been aided by considering evolutionarily coupled pairs22,23.\n\nA native contact map derived from a single native structure is often not sufficient to encode all the functionally relevant, minimally frustrated interactions. This led to the development of a variety of “multi-basin” models, where multiple experimental structures or structural constraints are included in a single SBM24–26. As described above, residue pairs with the highest DI scores, the high DI pairs (HDPs), are consistent with the native contact maps. Thus, in an analogous fashion, predicted contact constraints from co-evolution can be merged with contact maps computed from experimental structures in order to more fully capture the true underlying biomolecular energy landscape, including functional transitions and conformations, and therefore to be consistent with multiple structures27–29.\n\n\nRecent advances\n\nInteractions between proteins are fundamental to cellular processes. Where these interactions involve direct contact, multimeric structures, both long-lived and transient, leave correlated mutational patterns between interacting surface residues. A pioneering study used the HDPs between a histidine kinase and its response regulator to make a prediction of the transient protein complex enabling phosphotransfer30. This allowed a prediction for the histidine kinase TM0853 and its response regulator TM0468 that was later confirmed experimentally to be within 3.3 Å31. These predictions are made by minimizing a contact-based energy function consisting of dimeric HDPs. Where dimerization only weakly perturbs the monomer structure, refined rigid-body modeling in combination with co-evolutionary constrains can be employed to estimate protein complexes. When combined with experimental observations, directly coupled amino acids can unveil protein interfaces relevant for the study of disease32. Larger monomer distortions can be readily sampled with SBMs coupled with simulated annealing33. Current protocols involving HDPs have allowed the large-scale prediction of both homodimers34 and heterodimers35,36. The HDP contact map for a protein family that forms homodimers is a prime example of how ambiguity can arise in co-evolutionary information. The co-evolving dimeric interfacial contacts are mixed with HDPs selected for monomeric folding, but the dimeric contacts can in general be sorted from the monomeric contacts when there is a known monomer structure34. But rarely are there true dichotomies in biology; the existence of domain swapping37,38 and structural symmetry26,39 highlights some difficulties in assigning particular roles to each HDP. Also, some protein-protein interactions are mediated by disordered regions that order upon binding. The utility of DCA in these cases remains to be tested.\n\nIn addition to homo-multimerization, the set of conformations encoded in HDP contact maps can include functional motions. Multi-domain proteins can undergo conformational changes, for example, to accommodate ligands40 or in response to phosphorylation41. In periplasmic ligand binding proteins, there exists an open, ligand-free configuration and a closed, ligand-bound configuration. Molecular dynamics simulations can be performed by using an SBM specific to an open configuration but overlaid with an additional potential term consisting of a set of attractive, short-range interactions for each HDP27. Figure 1 illustrates an example of this for the leucine-binding protein. The native contact maps for two crystal structures of leucine-binding protein are shown in Figure 1A: “open” without ligand and “closed” with ligand. The closed contact map has additional contacts not present in the open structure. The DCA contact map, shown as the lower triangular map in Figure 1B, contains a superset of both the open and closed configuration contacts. An SBM is constructed that is specific to the open structure (Figure 1D) and additionally contains contact potentials stabilizing all the “non-native” DCA contacts (i.e., any DCA contacts that are not already in the open structure). These additional contacts are each given a stabilizing potential with a minimum at 8 Å. Molecular dynamics simulations of this hybrid SBM+DCA Hamiltonian show two clusters, each within 2 Å of either the open or closed state. Overlaying the DCA contacts does not disrupt the stability of the open structure, and additionally reveals the closed state without including any information from the closed crystal structure. This shows that co-evolutionary information can be used to uncover intermediary, hidden, and functionally relevant conformational states present in many protein families27.\n\n(A) Leucine-binding protein (LBP) contact maps derived from crystal structures: “open” without ligand and “closed” bound to a ligand. Each triangular region in the map shows a mark if residue pairs are less than 8 Å apart in the experimental structure. The closed contact map (upper triangle) has additional contacts not present in the open structure. (B) The DCA contact map inferred from residue co-evolution (lower triangle) contains a superset of contacts from both open and closed conformations. (C) A cartoon representation of the aligned open (apo) and closed (holo) LBP structures shows a large conformational change upon ligand binding. (D) A structure-based model (SBM) is defined from the apo structure plus contact potentials stabilizing DCA contacts that are not already in the open structure. A two-dimensional root mean square deviation (RMSD) distribution of the states explored by molecular dynamics simulations of this hybrid Hamiltonian shows two peaks within 2 Å of the open and closed states. This shows the ability to uncover functional states via co-evolutionary couplings.\n\nSo far, we have discussed how HDP contact maps can be used for structural modeling. However, the fundamental output of the DCA algorithm is not direct information about co-evolving pairs but rather a Potts model Hamiltonian describing the effective energies of interaction for all pairs of residues in a protein family. This Hamiltonian, though not transferable to any sequences outside the family, should, in principle, be able to provide a quantitative window into the stabilities provided by each amino acid in a protein. Strong evidence of the utility of the effective energies comes from their ability to predict the stability changes of single-site mutants42,43 and significant correlations to folding rates44. Including the so-called single-site fields in addition to the pairwise energies provides even better predictive power45. These results suggested that the pairwise energies calculated from co-evolution could be used to inform thermodynamic models of protein folding. Indeed, folding simulations using SBMs with DCA-weighted native contact potentials can better capture transition state ensembles46. DCA energies have also been shown to correlate with physical potentials when summed over the entire sequence47. Confidence in the ability to estimate energies at both the single-mutant and full-sequence levels is allowing novel methods for investigating the effective energy landscape of evolution, and bridging the gap between biophysics and sequence evolution47,48. These developments are important for integrating the energetics of protein folding and function with protein evolution and selection, which will be crucial to understanding drug resistance and cancer development going forward.\n\n\nFuture directions\n\nThe marriage between co-evolutionary information and physical models of biomolecules has been shown to be a fertile research field, where the most important results are yet to come. This field has been focused on rigorously validating the connection and usefulness between evolutionary information with structural modeling and experimental information. However, the true utility of co-evolutionary information is that it allows us to go places that are hard to access by current experimental technologies; important examples are those of membrane protein structure22,23 and dynamics, systems with transient conformational states, as well as investigation of large molecular assemblies that resist crystallographic characterization. Although crystal structures exist for FtsH AAA peptidase and the 30S ribosome, recent studies on these two systems28,49 show the promise of co-evolutionary information for discovering structural constraints in molecular assemblies. The ability to detect relevant evolutionary interactions has repercussions to our understanding of biomolecular assembly and function. Hopefully, these new tools can be used to alter protein conformation and rewire their interfaces. This has potential applications in the field of protein engineering, as well as systems biology. There is no conceptual hurdle to resisting the application of these ideas to RNA structure and function as well as protein-RNA interactions. Ultimately, we would hope to use all this knowledge to tackle biomedical problems that would help advance human health.\n\n\nAbbreviations\n\nDCA, direct coupling analysis; DI, direct information; HDP, high direct information pair; MSA, multiple sequence alignment; SBM, structure-based model.", "appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThis work was supported by the Center for Theoretical Biological Physics sponsored by the National Science Foundation (grants PHY-1427654 and NSF-MCB-1214457). Jeffrey K. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "12093", "date": "26 Jan 2016", "name": "Shoji Takata", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12094", "date": "26 Jan 2016", "name": "Angel Garcia", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12095", "date": "26 Jan 2016", "name": "Sudip Kundu", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-106
https://f1000research.com/articles/5-105/v1	26 Jan 16	{ "type": "Review", "title": "West Nile Virus Infection in the Central Nervous System", "authors": [ "Evandro R. Winkelmann", "Huanle Luo", "Tian Wang", "Evandro R. Winkelmann", "Huanle Luo" ], "abstract": "West Nile virus (WNV), a neurotropic single-stranded flavivirus has been the leading cause of arboviral encephalitis worldwide. Up to 50% of WNV convalescent patients in the United States were reported to have long-term neurological sequelae. Neither antiviral drugs nor vaccines are available for humans. Animal models have been used to investigate WNV pathogenesis and host immune response in humans. In this review, we will discuss recent findings from studies in animal models of WNV infection, and provide new insights on WNV pathogenesis and WNV-induced host immunity in the central nervous system.", "keywords": [ "West Nile Virus", "west nile virus infection in central nervous system", "central nervous system infection", "flavivirus", "arboviral encephalitis" ], "content": "Introduction\n\nWest Nile virus (WNV), a mosquito-borne, single-stranded, positive-sense flavivirus, has been the leading cause of viral encephalitis in the US for more than a decade. The virus was originally isolated from Uganda in 1937, later caused epidemic outbreaks in Asia, Europe, and Australia, and was introduced into the US in 19991. Human infection results mostly from mosquito bites, blood transfusion, organ transplantation, or occupational exposure2,3. In addition, WNV transmission through breastfeeding and in utero exposure is possible, although recent evidence suggests that the risk is very low4–7. About 80% of human infections with WNV are asymptomatic. The features of acute illness range from West Nile fever, to neuroinvasive conditions, including meningitis, encephalitis, acute flaccid paralysis, and death8. Up to 50% of convalescent patients with WNV have been reported to have long-term neurological sequelae or develop chronic kidney diseases, or both9–17. Although serologic and organ screening may reduce the risk of WNV infection through blood transfusion and organ transplantation18–20, there is no specific therapeutic agent for treatment of WNV infection, and an approved vaccine is not currently available for humans.\n\nAnimal models, which recapitulate WNV-induced neurological diseases in humans, have been effective in vivo experimental models to investigate WNV pathogenesis and host immune response21–23. In this review, we discuss recent findings from studies in animal models of WNV infection and provide new insights on WNV pathogenesis and virus-induced host immunity in the central nervous system (CNS).\n\n\nWest Nile virus entry into the central nervous system\n\nThe natural transmission of WNV in humans occurs through mosquito bites24. Keratinocytes and Langerhans cells (LCs) are the initial target cells where the virus is naturally deposited. WNV infection in keratinocytes induces innate cytokine responses mediated by Toll-like receptor (TLR) 7, which further promotes LC migration from the epidermis and accumulation in the local draining lymph nodes, where the virus is amplified before dissemination to kidney, spleen, and other visceral organs25–27. Following a systemic infection, WNV crosses the blood-brain barrier (BBB) after a brief viremia and ultimately invades the CNS28.\n\nThe development of WNV encephalitis is correlated with the ability of the virus to gain access to the CNS (neuroinvasiveness). At present, the mechanisms by which WNV enters the brain are not well understood. As a higher viral burden in serum usually correlates with earlier viral entry into the brain, it has been suggested that WNV infects the CNS in part via hematogenous spread29. The BBB is a complex structure that is composed of the tight endothelium formed by endothelial cells through tight junctions and smooth muscle cells surrounded by a layer of astrocytic foot processes30,31. Systemic WNV replication-induced innate cytokine responses are known to control BBB integrity (Table 1). Pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and macrophage migration inhibitory factor (MIF), contribute to the disruption of the BBB32–34. In addition, matrix metalloproteinase 9 (MMP9), which is upregulated upon WNV infection in both the periphery and mouse brain, facilitates WNV entry into the brain by enhancing BBB permeability35. Semaphorin 7A (Sema7A), a potent stimulator of monocytes and neutrophils, acts upstream of the host inflammatory reaction during WNV infection. Following infection, Sema7A-deficient mice produced less TNF-α in the periphery and had a reduced BBB permeability compared with wild-type controls36. In contrast to the effects of pro-inflammatory cytokines, both type I interferon (IFN) (IFN-α and IFN-β) and type III (IFN-λ) are implicated in promoting BBB integrity. Daniels et al. have recently demonstrated that type I IFNs play a direct role in endothelial permeability and tight junction formation via balanced activation of the small guanosine triphosphatases (GTPases) Rac1 and RhoA interactions and indirect suppression of the effects of TNF-α and IL-1β33. The TAM receptors Tyro3, Axl, and Mertk are receptor tyrosine kinases that dampen host innate immune responses upon interactions with their ligands Gas6 and Protein S, which recognize phosphatidylserine on apoptotic cells37. A recent study showed that activation of Mertk synergized with IFN-β to tighten cell junctions and prevent virus transit across brain microvascular endothelial (BMVE) cells. As a consequence, mice lacking Mertk or Axl (or both), but not Tyro3, exhibited greater vulnerability to infection with neuroinvasive WNV38. In another study39, mice lacking IFN-λ signaling were shown to have increased viral titers in the brain and spinal cord during WNV infection. This is not associated with a direct antiviral effect of IFN-λ in the CNS. Instead, IFN-λ signaling in BMVE cells modulates tight junction protein localization in a protein synthesis- and signal transducer and activator of transcription 1 (STAT1)-independent manner, which increases transendothelial electrical resistance and decreases virus movement across the BBB. Besides innate cytokines, upregulation of cell adhesion molecules (CAMs), such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and E-selectin expression on WNV-infected BMVEs induces the adhesion and transendothelial migration of leukocytes across these cells40,41 (Table 1). Moreover, direct infection of BMVE cells facilitates the entry of cell-free virus into the CNS without disturbing the BBB, and an increase on CAM further assists the trafficking of WNV-infected immune cells into the CNS via a ‘Trojan horse’ mechanism, thereby contributing to WNV dissemination into the CNS and its associated pathology42–44. Finally, infection of the olfactory neurons and consequent dissemination to the olfactory bulb and direct axonal retrograde transport of virus that has infected peripheral neurons have been postulated as the other mechanisms of WNV entry into the CNS45–47.\n\nBBB, blood-brain barrier; CAM, Cell adhesion molecules; ICAM-1, intercellular adhesion molecule-1; IFN, interferon; IL-1β, interleukin-1 beta; MIF, macrophage migration inhibitory factor; TNF-α, tumor necrosis factor-alpha; VCAM-1, vascular cell adhesion molecule-1.\n\nThe host has developed multiple strategies to limit virus dissemination in the periphery and prevent the trafficking of WNV across the BBB. First, WNV infection activates the signaling pathways of several pattern recognition receptors (PRRs), including TLRs 3 and 7 and RIG-I-like receptor (RLR), in order to boost innate immunity, culminating in the synthesis of antiviral cytokines, such as type I IFNs and pro-inflammatory cytokines34,48–50. Type 1 IFNs and IFN-stimulating genes (ISGs) both participate in the control of viral infections and prevent WNV from invading the CNS51–54. Next, innate immune cells such as γδ T cells (in particular, the Vγ1+ cell subpopulation) expand significantly during WNV infection and play a role in the early control of WNV dissemination mainly through the secretion of IFN-γ55–57. These cells are also involved in the regulation of adaptive immunity against WNV infection and persistence, presumably mediated by the crosstalk between γδ T cells and dendritic cells (DCs), either via direct contact or indirectly through secreting molecules, to promote DC maturation and activation and ultimately to help T cell priming during WNV infection58. TCRδ–/– mice (γδ T cell-deficient mice) were shown to have a numeric and functional reduction in memory T cell responses to WNV infection compared with wild-type mice59. Furthermore, B cells and specific antibodies are critical in the control of disseminated WNV infection but are not sufficient to eliminate it from the host29,60–62. In particular, induction of a specific, neutralizing IgM response early during infection limits viremia and dissemination into the CNS and protects the host against lethal infection29. In addition, the complement system has been shown to control WNV infection, in part through its ability to induce a protective antibody response and by priming adaptive immune responses through distinct mechanisms63,64. αβ T cells61 provide long-lasting protective immunity and contribute to host survival following WNV infection. Among them, CD4+ T cells respond vigorously in the periphery65, whereas CD8+ T cell responses have been observed in both the spleen and brain following WNV infection66. CD4+ T cells provide help for antibody responses and sustain WNV-specific CD8+ T cell responses in the tissues that enable viral clearance67. CD8+ T cell responses are critical in clearing WNV infection from tissues and preventing viral persistence68. Furthermore, higher levels of peripheral regulatory T cells (Tregs) after infection are known to protect against severe WNV disease in immunocompetent animals and humans69. Tregs are also critical in generating a pool of WNV-specific memory T cells in the periphery70. RLR-mediated innate signaling is involved in regulating adaptive immunity against pathogenic WNV. Mice deficient in mitochondrial antiviral signaling protein (MAVS), the adaptor protein for RLR signaling, were reported to exhibit lower neutralization ability of WNV-specific antibodies and increased numbers of virus-specific CD8+ T cells, with non-specific immune cell proliferation in the periphery71.\n\nAging is a known risk factor for WNV-induced encephalitis23,72,73. The decline in immunity seen in the elderly is a significant contributor to the increased risk of infection. In an aged mouse model of WNV infection, Vγ1+ cells displayed a slower, reduced response to WNV infection compared with those of young adult mice57. Although the young adult CD4+ or CD8+ T cells readily protected immunodeficient mice upon WNV infection, T cells of either subset in aged mice were unable to provide WNV-specific protection74. Furthermore, a recent study75 reported that age-dependent cell-intrinsic and environmental defects in the draining lymph nodes result in delayed immune cell recruitment and antigen recognition by CD4+ T cells, leading to impaired IgM and IgG and increased susceptibility to WNV infection in old mice.\n\n\nWest Nile virus infection and West Nile virus-induced immune responses in the central nervous system\n\nOnce in the brain, WNV can infect and replicate in various types of CNS residential cells, including neurons, astrocytes, and microglial cells76,77. In a recent study, by using a spinal cord slice culture (SCSC) model, CNS-resident cells were demonstrated to have the capacity to initiate a robust innate immune response against WNV infection in the absence of infiltrating inflammatory cells and systemic immune responses76. CNS cells display differential susceptibility to WNV infection, dependent on their cell-intrinsic host defense programs and cellular activities (Table 2). The antiviral action of ISG-Ifit2 restricts WNV spread in the CNS, especially during the early stages of virus spread78. Granule cell neurons (GCNs) of the cerebellum, which comprise the largest population of neurons in the brain, express high levels of genes associated with the host defense pathway, including a STAT1- and IFN-dependent signaling signature both at the basal level and after IFN-β treatment. These cells are less susceptible than cortical neurons to WNV replication79. Multiple PRR pathways are involved in the induction of antiviral responses in neurons. IL-1β production triggered by non-obese diabetic (NOD)-like receptor family pyrin domain containing 3 (NLRP3)-inflammasome suppressed WNV replication in neurons80. Both myeloid differentiation primary response gene 88 (MyD88)- and TLR3-mediated innate signaling play protective roles against WNV infection, in part by inhibiting replication in subsets of neurons81,82. Caspase-12 controls WNV infection in neurons by regulating E3 ubiquitin ligase TRIM25-mediated ubiquitination of RIG-I, which induces high levels of IFN responses83. WNV also triggers production of TNF-α, IL-6, and IFN-β in microglia in a TLR3-dependent manner34,84. In astrocytes, cellular activities such as inhibiting high levels of furin-like protease activity contribute to a reduced susceptibility to the avirulent WNV-MAD78 infection85. WNV infection in CNS cells also triggers the secretion of chemokines, which further facilitates immune cells to cross the BBB. For example, WNV-specific CXCR3+CD8+ T cells preferred to move into the cerebellum, where WNV-infected GCNs expressed a high grade of CXCL10 to clear virus86. Accordingly, the CXCL10-deficient mice had decreased CD8+ T cell infiltration and increased viral load in the brain87. Likewise, the expression of chemokine receptor CCR5 and its ligand CCL5 was prominently upregulated by WNV infection in the CNS. CCR5 has been implicated for the recruitment of leukocytes (including CD4+ and CD8+ T cells, natural killer cells, and macrophages) into the brain. CCR5-deficient mice rapidly succumbed to WNV infection and were not able to clear the virus in the brain during the time course of the disease88.\n\nCNS, central nervous system; IFN, interferon; IL, interleukin; TLR, Toll-like receptor; TNF-α, tumor necrosis factor-alpha; WNV, West Nile virus.\n\nUnder normal physiological conditions, the integrity of the BBB restricts the infiltration of leukocytes and maintains the CNS as an immune-specialized compartment. Following systemic WNV infection, immune responses in the CNS are induced by infiltrating inflammatory cells, including microglia/macrophages, neutrophils, and effector CD4+ and CD8+ T cells (Table 2)67,88–91, and further infection in the CNS residential cells. Monocyte infiltration into the CNS is the hallmark of WNV encephalitis. After entry into the CNS, they differentiate into macrophages and microglia88,92. Monocytes are presumably protective against WNV infection, as depletion of these cells increased mortality of mice infected with a neurotropic strain of WNV93. However, inhibition of Ly6Chi monocyte trafficking into the brain by anti-very late antigen-4 (anti-VLA-4) integrin antibody blockade at the time of observation of the first weight loss and leukocyte influx resulted in long-term survival in mice with lethal encephalitis94. Although CCR2, a receptor expressed on Ly6Chi inflammatory monocytes, is not directly involved in the recruitment of these cells from blood to the brain, it promotes peripheral monocytosis during WNV infection, which is critical for ultimate accumulation of monocytes in the brain, and host protection95. It was demonstrated that the CCR2 chemokine ligands CCL2 and CCL7 are involved in the monocytosis and monocyte accumulation in the brain. The deficiency of CCL7 in mice leads to increased WNV load in the brain and enhanced mortality, suggesting a critical role of CCL7-mediated immune response in host protection96. Furthermore, TLR7- and IL-23-dependent immune responses were known to mediate CD11b+ macrophage migration into the CNS and protect the host from lethal WNV infection49. Neutrophils are another cell type detected in the CNS, but the role of these cells in WNV infection has yet to be elucidated. Evaluation of cell infiltrate in cerebrospinal fluid from human cases of WNV meningitis and encephalitis showed high counts of neutrophils97,98. Neutrophils were reported to display dual functions in WNV-infected mice. They could serve as reservoirs for virus replication in the periphery at early stage of infection, but are involved in viral clearance at the late stage of infection in the CNS43. IL-22, a cytokine implicated in the modulation of the chemokine receptor CXCR2, is responsible for the recruitment of neutrophils99. Finally, CD4+ T cells are required for the clearance of WNV from CNS and therefore mice survival, providing help for antibody responses, and sustaining WNV-specific CD8+ T cell responses in the CNS that enable viral clearance67. They also have a direct antiviral role in the CNS by producing IFN-γ and IL-2 and have a potential for in vivo and ex vivo cytotoxicity100. It has been demonstrated that IL-1R1 signaling modulates CD11c+ cell-mediated T cell reactivation and promotes virologic control during WNV infection within the CNS101. CD8+ T cells have an important function in clearing infection from the CNS and preventing viral persistence upon a low-dose WNV challenge68,102,103. Nevertheless, CD8+ T cells are also involved in promoting CNS pathology following high doses of WNV infection104,105. Thus, depending on the viral dosage, CD8+ T cells could be involved in both recovery and immunopathology in WNV infection. The effector function of T cells to control virus replication and eliminate the infection is directed through the production of cytokines, including IFN-γ, or direct killing of infected cells through secretion of perforin and signaling through Fas to -Fas ligand, or TNF-related apoptosis-inducing ligand (TRAIL)-dependent pathways68,102,103. RLR-mediated innate signaling was reported to shape optimal CD8+ T cell activation and subsequent clearance of WNV from the CNS106. CD11b+CD45hi infiltrating cells (macrophages) are the primary producers of IL-1β within the CNS. By using an in vitro BBB model, Durrant et al. have shown that IL-1β promotes CXCR4-mediated CD8+ T lymphocyte adhesion to BMVE cells. Furthermore, inhibition of CXCR4 promotes T lymphocyte entry into the CNS parenchyma, and this increases viral clearance and ultimately improves survival and reduces viral loads107. Thus, perivascular localization might hinder antiviral immune responses during WNV encephalitis.\n\n\nConclusions\n\nIn summary, following systemic WNV infection, immune responses in the periphery help to control virus dissemination, whereas CNS immunity is critical for the clearance of virus. WNV has been the leading cause of viral encephalitis in the US for more than a decade. Current efforts on drug development have been focused mostly on the inhibitors of virus replication. Results from animal studies will provide a better understanding of the mechanisms by which flaviviruses enter the brain and induce lethal encephalitis; they may also lead to the development of new strategies to prevent and treat WNV-induced encephalitis.", "appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nThis work was supported in part by National Institutes of Health grant R01 AI099123 (TW).\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nNash D, Mostashari F, Fine A, et al.: The outbreak of West Nile virus infection in the New York City area in 1999. N Engl J Med. 2001; 344(24): 1807–14. PubMed Abstract \| Publisher Full Text\n\nCenters for Disease Control and Prevention (CDC): Laboratory-acquired West Nile virus infections--United States, 2002. MMWR Morb Mortal Wkly Rep. 2002; 51(50): 1133–5. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMonath TP, Cropp CB, Harrison AK: Mode of entry of a neurotropic arbovirus into the central nervous system. Reinvestigation of an old controversy. Lab Invest. 1983; 48(4): 399–410. PubMed Abstract\n\nErrett JS, Suthar MS, McMillan A, et al.: The essential, nonredundant roles of RIG-I and MDA5 in detecting and controlling West Nile virus infection. J Virol. 2013; 87(21): 11416–25. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTown T, Bai F, Wang T, et al.: Toll-like receptor 7 mitigates lethal West Nile encephalitis via interleukin 23-dependent immune cell infiltration and homing. Immunity. 2009; 30(2): 242–53. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nFredericksen BL, Keller BC, Fornek J, et al.: Establishment and maintenance of the innate antiviral response to West Nile Virus involves both RIG-I and MDA5 signaling through IPS-1. J Virol. 2008; 82(2): 609–16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKatze MG, He Y, Gale M Jr: Viruses and interferon: a fight for supremacy. Nat Rev Immunol. 2002; 2(9): 675–87. PubMed Abstract \| Publisher Full Text\n\nSamuel MA, Diamond MS: Alpha/beta interferon protects against lethal West Nile virus infection by restricting cellular tropism and enhancing neuronal survival. J Virol. 2005; 79(21): 13350–61. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLazear HM, Pinto AK, Vogt MR, et al.: Beta interferon controls West Nile virus infection and pathogenesis in mice. J Virol. 2011; 85(14): 7186–94. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThackray LB, Shrestha B, Richner JM, et al.: Interferon regulatory factor 5-dependent immune responses in the draining lymph node protect against West Nile virus infection. J Virol. 2014; 88(19): 11007–21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang T, Scully E, Yin Z, et al.: IFN-gamma-producing gamma delta T cells help control murine West Nile virus infection. J Immunol. 2003; 171(5): 2524–31. PubMed Abstract \| Publisher Full Text\n\nShrestha B, Wang T, Samuel MA, et al.: Gamma interferon plays a crucial early antiviral role in protection against West Nile virus infection. J Virol. 2006; 80(11): 5338–48. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWelte T, Lamb J, Anderson JF, et al.: Role of two distinct gammadelta T cell subsets during West Nile virus infection. FEMS Immunol Med Microbiol. 2008; 53(2): 275–83. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFang H, Welte T, Zheng X, et al.: gammadelta T cells promote the maturation of dendritic cells during West Nile virus infection. FEMS Immunol Med Microbiol. 2010; 59(1): 71–80. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang T, Gao Y, Scully E, et al.: Gamma delta T cells facilitate adaptive immunity against West Nile virus infection in mice. J Immunol. 2006; 177(3): 1825–32. PubMed Abstract \| Publisher Full Text\n\nDiamond MS, Shrestha B, Marri A, et al.: B cells and antibody play critical roles in the immediate defense of disseminated infection by West Nile encephalitis virus. J Virol. 2003; 77(4): 2578–86. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDiamond MS, Shrestha B, Mehlhop E, et al.: Innate and adaptive immune responses determine protection against disseminated infection by West Nile encephalitis virus. Viral Immunol. 2003; 16(3): 259–78. PubMed Abstract \| Publisher Full Text\n\nRoehrig JT, Staudinger LA, Hunt AR, et al.: Antibody prophylaxis and therapy for flavivirus encephalitis infections. Ann N Y Acad Sci. 2001; 951: 286–97. PubMed Abstract \| Publisher Full Text\n\nMehlhop E, Diamond MS: Protective immune responses against West Nile virus are primed by distinct complement activation pathways. J Exp Med. 2006; 203(5): 1371–81. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMehlhop E, Whitby K, Oliphant T, et al.: Complement activation is required for induction of a protective antibody response against West Nile virus infection. J Virol. 2005; 79(12): 7466–77. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKulkarni AB, Müllbacher A, Blanden RV: Functional analysis of macrophages, B cells and splenic dendritic cells as antigen-presenting cells in West Nile virus-specific murine T lymphocyte proliferation. Immunol Cell Biol. 1991; 69(Pt 2): 71–80. PubMed Abstract \| Publisher Full Text\n\nLiu Y, Blanden RV, Müllbacher A: Identification of cytolytic lymphocytes in West Nile virus-infected murine central nervous system. J Gen Virol. 1989; 70(Pt 3): 565–73. PubMed Abstract \| Publisher Full Text\n\nSitati EM, Diamond MS: CD4+ T-cell responses are required for clearance of West Nile virus from the central nervous system. J Virol. 2006; 80(24): 12060–9. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nShrestha B, Diamond MS: Role of CD8+ T cells in control of West Nile virus infection. J Virol. 2004; 78(15): 8312–21. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nLanteri MC, O'Brien KM, Purtha WE, et al.: Tregs control the development of symptomatic West Nile virus infection in humans and mice. J Clin Invest. 2009; 119(11): 3266–77. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nGraham JB, Da Costa A, Lund JM: Regulatory T cells shape the resident memory T cell response to virus infection in the tissues. J Immunol. 2014; 192(2): 683–90. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nSuthar MS, Ma DY, Thomas S, et al.: IPS-1 is essential for the control of West Nile virus infection and immunity. PLoS Pathog. 2010; 6(2): e1000757. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nCampbell GL, Marfin AA, Lanciotti RS, et al.: West Nile virus. Lancet Infect Dis. 2002; 2(9): 519–29. PubMed Abstract \| Publisher Full Text\n\nSolomon T, Ooi MH, Beasley DW, et al.: West Nile encephalitis. BMJ. 2003; 326(7394): 865–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrien JD, Uhrlaub JL, Hirsch A, et al.: Key role of T cell defects in age-related vulnerability to West Nile virus. J Exp Med. 2009; 206(12): 2735–45. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nRichner JM, Gmyrek GB, Govero J, et al.: Age-Dependent Cell Trafficking Defects in Draining Lymph Nodes Impair Adaptive Immunity and Control of West Nile Virus Infection. PLoS Pathog. 2015; 11(7): e1005027. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nQuick ED, Leser JS, Clarke P, et al.: Activation of intrinsic immune responses and microglial phagocytosis in an ex vivo spinal cord slice culture model of West Nile virus infection. J Virol. 2014; 88(22): 13005–14. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nShrestha B, Gottlieb D, Diamond MS: Infection and injury of neurons by West Nile encephalitis virus. J Virol. 2003; 77(24): 13203–13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCho H, Shrestha B, Sen GC, et al.: A role for Ifit2 in restricting West Nile virus infection in the brain. J Virol. 2013; 87(15): 8363–71. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nCho H, Proll SC, Szretter KJ, et al.: Differential innate immune response programs in neuronal subtypes determine susceptibility to infection in the brain by positive-stranded RNA viruses. Nat Med. 2013; 19(4): 458–64. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nRamos HJ, Lanteri MC, Blahnik G, et al.: IL-1β signaling promotes CNS-intrinsic immune control of West Nile virus infection. PLoS Pathog. 2012; 8(11): e1003039. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nDaffis S, Samuel MA, Suthar MS, et al.: Toll-like receptor 3 has a protective role against West Nile virus infection. J Virol. 2008; 82(21): 10349–58. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nSzretter KJ, Daffis S, Patel J, et al.: The innate immune adaptor molecule MyD88 restricts West Nile virus replication and spread in neurons of the central nervous system. J Virol. 2010; 84(23): 12125–38. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nWang P, Arjona A, Zhang Y, et al.: Caspase-12 controls West Nile virus infection via the viral RNA receptor RIG-I. Nat Immunol. 2010; 11(10): 912–9. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nTown T, Jeng D, Alexopoulou L, et al.: Microglia recognize double-stranded RNA via TLR3. J Immunol. 2006; 176(6): 3804–12. PubMed Abstract \| Publisher Full Text\n\nHussmann KL, Samuel MA, Kim KS, et al.: Differential replication of pathogenic and nonpathogenic strains of West Nile virus within astrocytes. J Virol. 2013; 87(5): 2814–22. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang B, Chan YK, Lu B, et al.: CXCR3 mediates region-specific antiviral T cell trafficking within the central nervous system during West Nile virus encephalitis. J Immunol. 2008; 180(4): 2641–9. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nKlein RS, Lin E, Zhang B, et al.: Neuronal CXCL10 directs CD8+ T-cell recruitment and control of West Nile virus encephalitis. J Virol. 2005; 79(17): 11457–66. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nGlass WG, Lim JK, Cholera R, et al.: Chemokine receptor CCR5 promotes leukocyte trafficking to the brain and survival in West Nile virus infection. J Exp Med. 2005; 202(8): 1087–98. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nBréhin AC, Mouriès J, Frenkiel MP, et al.: Dynamics of immune cell recruitment during West Nile encephalitis and identification of a new CD19+B220-BST-2+ leukocyte population. J Immunol. 2008; 180(10): 6760–7. PubMed Abstract \| Publisher Full Text\n\nLim JK, Obara CJ, Rivollier A, et al.: Chemokine receptor Ccr2 is critical for monocyte accumulation and survival in West Nile virus encephalitis. J Immunol. 2011; 186(1): 471–8. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nSitati E, McCandless EE, Klein RS, et al.: CD40-CD40 ligand interactions promote trafficking of CD8+ T cells into the brain and protection against West Nile virus encephalitis. J Virol. 2007; 81(18): 9801–11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGetts DR, Terry RL, Getts MT, et al.: Ly6c+ \"inflammatory monocytes\" are microglial precursors recruited in a pathogenic manner in West Nile virus encephalitis. J Exp Med. 2008; 205(10): 2319–37. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nBen-Nathan D, Huitinga I, Lustig S, et al.: West Nile virus neuroinvasion and encephalitis induced by macrophage depletion in mice. Arch Virol. 1996; 141(3–4): 459–69. PubMed Abstract \| Publisher Full Text\n\nGetts DR, Terry RL, Getts MT, et al.: Targeted blockade in lethal West Nile virus encephalitis indicates a crucial role for very late antigen (VLA)-4-dependent recruitment of nitric oxide-producing macrophages. J Neuroinflammation. 2012; 9: 246. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nLim JK, Obara CJ, Rivollier A, et al.: Chemokine receptor Ccr2 is critical for monocyte accumulation and survival in West Nile virus encephalitis. J Immunol. 2011; 186(1): 471–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBardina SV, Michlmayr D, Hoffman KW, et al.: Differential Roles of Chemokines CCL2 and CCL7 in Monocytosis and Leukocyte Migration during West Nile Virus Infection. J Immunol. 2015; 195(9): 4306–18. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nRawal A, Gavin PJ, Sturgis CD: Cerebrospinal fluid cytology in seasonal epidemic West Nile virus meningo-encephalitis. Diagn Cytopathol. 2006; 34(2): 127–9. PubMed Abstract \| Publisher Full Text\n\nTyler KL, Pape J, Goody RJ, et al.: CSF findings in 250 patients with serologically confirmed West Nile virus meningitis and encephalitis. Neurology. 2006; 66(3): 361–5. PubMed Abstract \| Publisher Full Text\n\nWang P, Bai F, Zenewicz LA, et al.: IL-22 signaling contributes to West Nile encephalitis pathogenesis. PLoS One. 2012; 7(8): e44153. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrien JD, Uhrlaub JL, Nikolich-Zugich J: West Nile virus-specific CD4 T cells exhibit direct antiviral cytokine secretion and cytotoxicity and are sufficient for antiviral protection. J Immunol. 2008; 181(12): 8568–75. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDurrant DM, Robinette ML, Klein RS: IL-1R1 is required for dendritic cell-mediated T cell reactivation within the CNS during West Nile virus encephalitis. J Exp Med. 2013; 210(3): 503–16. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nShrestha B, Pinto AK, Green S, et al.: CD8+ T cells use TRAIL to restrict West Nile virus pathogenesis by controlling infection in neurons. J Virol. 2012; 86(17): 8937–48. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nShrestha B, Samuel MA, Diamond MS: CD8+ T cells require perforin to clear West Nile virus from infected neurons. J Virol. 2006; 80(1): 119–29. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSzretter KJ, Daniels BP, Cho H, et al.: 2'-O methylation of the viral mRNA cap by West Nile virus evades ifit1-dependent and -independent mechanisms of host restriction in vivo. PLoS Pathog. 2012; 8(5): e1002698. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang Y, Lobigs M, Lee E, et al.: CD8+ T cells mediate recovery and immunopathology in West Nile virus encephalitis. J Virol. 2003; 77(24): 13323–34. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nLazear HM, Pinto AK, Ramos HJ, et al.: Pattern recognition receptor MDA5 modulates CD8+ T cell-dependent clearance of West Nile virus from the central nervous system. J Virol. 2013; 87(21): 11401–15. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nDurrant DM, Daniels BP, Klein RS: IL-1R1 signaling regulates CXCL12-mediated T cell localization and fate within the central nervous system during West Nile Virus encephalitis. J Immunol. 2014; 193(8): 4095–106. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation" }	[ { "id": "11967", "date": "26 Jan 2016", "name": "Sebastian Ulbert", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "11969", "date": "26 Jan 2016", "name": "Giorgio Palù", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "11971", "date": "26 Jan 2016", "name": "Anna Papa", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-105
https://f1000research.com/articles/5-104/v1	26 Jan 16	{ "type": "Review", "title": "An update around the evidence base for the lower extremity ultrasound regional block technique", "authors": [ "Andrea Fanelli", "Daniela Ghisi", "Rita Maria Melotti", "Daniela Ghisi", "Rita Maria Melotti" ], "abstract": "Ultrasound guidance currently represents the gold standard for regional anesthesia. In particular for lower extremity blocks, despite the heterogeneity and the lack of large randomized controlled trials, current literature shows a modest improvement in block onset and quality compared with other localization techniques. This review aims to present the most recent findings on the application of ultrasound guidance for each single lower extremity approach.", "keywords": [ "Ultrasound", "lower extremity blocks", "lumbar plexus", "sacral plexus" ], "content": "Introduction\n\nThe use of ultrasound applied to peripheral nerve blocks has been the object of several randomized controlled trials (RCTs) and reviews in the last two decades1–3. Despite the heterogeneity of RCTs comparing ultrasound guidance to other localization techniques in terms of type of block, anesthetic agents, and control groups, a recent systematic review published by Liu has shown a moderate superiority of ultrasound guidance for the majority of the evaluated block characteristics2. The effectiveness of ultrasound guidance showed by Liu was previously pointed out by Lewis et al.4. The authors conducted a meta-analysis showing that ultrasound guidance produces a superior success rate in terms of readiness for surgery after sensory and motor test and fewer blocks requiring analgesic supplementation or conversion to general anesthesia compared to other localization techniques4. Unfortunately, only six of the 32 studies considered by Lewis et al. were related to lower extremity blocks4.\n\nThe interest and the evidence related to the use of ultrasound guidance applied to lower extremity blocks have been growing in the last few years2. In fact, Liu2 identified nine new RCTs evaluating lower extremity blocks, beside the eight pre-existing studies included in the first review in 20105. Based on the analyzed data, the author shows that ultrasound guidance provides a modest improvement in block onset and quality when applied to lower extremity blocks2. In particular, three studies reported a faster onset by 5 to 14 minutes if ultrasound was used to perform lower extremity blocks2.\n\nThis review aims to present the most recent findings on the application of ultrasound guidance for main lower extremity blocks and approaches.\n\n\nAnatomy of interest for lower extremity peripheral nerve blocks\n\nUltrasound provides the possibility to directly visualize the nerves, vessels, needle, and local anesthetic distribution in the majority of patients6, but it does not replace the full comprehension of the anatomy that still represents the foundation for safe and successful blocks and for the right management of the anesthetic/analgesic plan.\n\nThe lumbar plexus is an anastomotic complex formed by the anterior branches from L1 to L4 roots7. The lumbosacral trunk is formed by the anterior branch of L5 together with an anastomotic branch from L4. The lumbar plexus has a triangular form: its base is represented by the lumbar vertebrae and its apex is formed by the union of the third roots with the ascendant rami of the fourth7. The lumbar plexus lies anterior to the transverse process within the psoas muscle. All of the branches of the lumbar plexus emerge from the psoas muscle and leave the pelvis. The lumbar plexus gives origin to ﬁbers for the main trunks of the lower extremity: the femoral and obturator nerves, as well as for the sciatic nerve through the lumbosacral trunk7.\n\nThe obturator nerve is formed by the L2−L4 roots. It leaves the psoas muscle at the level of the sacroiliac joint. In the pelvis, it runs close to the ureter and the internal iliac artery with an outward and downward direction. The obturator nerve leaves the pelvis and enters the thigh at the upper part of the obturator foramen, reaching the obturator groove, where in the majority of cases it divides into an anterior and a posterior branch7. The anterior branch runs in front of the adductor brevis and adductor magnus muscles and behind the adductor longus. The posterior branch runs between the adductor brevis and adductor magnus muscles7.\n\nThe femoral nerve is the main terminal branch of the lumbar plexus and is formed by the roots of L2 to L47. After its origin, the femoral nerve runs in the iliopsoas groove and enters the thigh toward the inguinal ligament, where it generally divides into its terminal branches7. In the femoral triangle, the nerve is localized laterally to the artery, deep to the fascia lata and to the fascia iliaca, and on the anterior aspect of the iliopsoas muscle7. The saphenous nerve is a terminal branch of the femoral and it is of clinical interest for knee and leg surgeries8. At the femoral triangle, the saphenous nerve is located within the posterior plane, and it innervates the skin of the medial part of the knee and the anteromedial part of the leg and foot. The saphenous nerve is also involved in the innervation of the knee joint7.\n\nThe sacral plexus is formed by the anterior sacral roots from S1 to S3 and by the lumbosacral trunk7. The sacral plexus transverses the sciatic foramen lying anterior to the piriformis muscle. At this level, it is separated from the visceral structures of the pelvis by the pelvic aponeurosis. In the parasacral region, the sacral plexus provides two branches involved in the innervation of the hip joint, potentially relevant for the anesthesia and analgesia of patients undergoing complex hip surgeries7. In the thigh, the sciatic nerve runs between the semitendinosus and semimembranosus muscles medially and the biceps femoris muscle laterally7. From the middle of the thigh to the popliteal crease, the common peroneal and tibial nerves give off branches to the posterior part of the knee joint. Inside the popliteal fossa, the tibial and common peroneal nerves provide a medial and a lateral sural cutaneous nerve respectively7.\n\nIn terms of nerve anatomy, the ratio of connective and neural tissue in the sciatic nerve increases from proximal to distal, to the point of creating a thick paraneural sheath around the nerve2,9. This sheath plays an important functional role in the spread and clinical effect of the local anesthetic injected during a sciatic block.\n\n\nLumbar plexus block\n\nThe application of ultrasound guidance for the lumbar plexus block is still challenging due to the depth of the nerve structures and the presence of the ‘acoustic shadow’ of the transverse processes10,11. In clinical practice, ultrasound guidance is commonly used for the lumbar plexus block in combination with nerve stimulation, and it can be applied either for pre-procedural scanning or as real-time guidance. Sonoanatomical studies have shown that it is possible to visualize the transverse processes, vertebral body, psoas major muscle, erector spinae, quadratus lumborum, lower pole of the kidney, peritoneum, aorta, and vena cava12,13. As previously described, the continuous psoas compartment block can be performed under real-time ultrasound guidance using either a longitudinal or a transversal scan and an in-plane needle approach1. When a longitudinal scan is preferred, a curved array transducer is used to perform the first subcostal scan to localize the inferior kidney pole. The second scan is dedicated to visualizing the sacral promontory in long axis as a sonoanatomical landmark, which allows one to ascend cranially to L2–L3, counting the shadows of the transverse processes14. A reliable catheterization of the lumbar plexus can be achieved after identifying the transverse processes of L2–L3 and the psoas muscle lying between them1. These sonoanatomical landmarks are always reproducible, whereas visualization of the lumbar nerve roots might be challenging with both transverse and longitudinal approaches. The advantage of the technique is that the needle is inserted in-plane, on a sagittal plane, to avoid any medial direction and potential epidural localization of the catheter, even if repeated needle contact with transverse processes could represent a drawback of the technique. The aid of a nerve stimulator can help in improving the reliability of the described approach. Recently, Karmakar et al. published a prospective case series evaluating the feasibility of a modified transverse scan of the lumbar paravertebral region with the ultrasound beam being insonated through the lumbar intertransverse space and directed medially toward the intervertebral foramen15. The target vertebral level for the lumbar plexus block is identified by locating the lumbosacral junction on a paramedian sagittal scan and then counting cranially to locate the lamina and transverse processes of the L3, L4, and L5 vertebrae. The modified approach introduced by Karmakar et al. is then performed with the transducer positioned 4 cm lateral to the midline in the transverse orientation at the L3−L4 intervertebral level15. The aim is to identify the lumbar nerve root close to the intervertebral foramen. The needle is inserted in-plane at a point 4 cm lateral to the midline and medial to the transducer and then it is slowly advanced under ultrasound guidance to the posterior aspect of the psoas muscle until either needle–lumbar plexus contact is visualized or an ipsilateral quadriceps muscle contraction is elicited15.\n\n\nSuprasacral parallel shift block\n\nAs reported by Bendtsen et al., anesthesia and analgesia for hip surgeries can be obtained with a single injection performed under ultrasound guidance reaching the terminal nerves of the lumbar plexus and the lumbosacral trunk; this new approach was denominated by the authors the suprasacral parallel shift block16. The insertion site of the needle is in correspondence of the interspace between the upper rim of the sacral ala and the lower border of the transverse process of L5. The local anesthetic solution has to be injected into the compartment behind the psoas muscle where the lumbosacral trunk, the obturator, and the femoral nerve are located. To perform the suprasacral parallel shift block, a curved array probe (2−5 MHz) is required and has to be initially placed parallel to the iliac crest and then moved medially until the sacral bone is recognized. Afterwards, the probe has to be rotated until the upper margin of the sacral bone, the transverse process of L5, and the interspace between the two bony structures come into view. The needle is inserted with an out-of-plane approach perpendicular to the skin and advanced until it penetrates the lumbosacral ligament and loss-of-resistance is perceived16.\n\nThe ultrasound-guided suprasacral parallel shift block was recently tested in terms of effectiveness for anesthesia of the terminal nerves of the lumbar plexus compared with a classic lumbar plexus block17. Moreover, the effectiveness of the two approaches to obtain anesthesia of the lumbosacral trunk was evaluated. This RCT conducted on volunteers showed that the suprasacral parallel shift block is at least as effective as the lumbar plexus block for blockade of the terminal nerves of the lumbar plexus and is significantly more effective for blockade of the lumbosacral trunk17. Unfortunately, the suprasacral parallel shift block is not superior to the lumbar plexus technique to provide anesthesia of all dermatomes from L2 to S117.\n\n\nFascia iliaca compartment block\n\nThe fascia iliaca compartment block, first described by Dalens et al.18, has been reported to effectively block the three major terminal nerves of the lumbar plexus: the femoral, the obturator, and the lateral cutaneous nerves. The fascia iliaca block has been the object of attention in the last few years due to its potential role as first-line pain therapy for patients presenting to the emergency department with a proximal femoral fracture19. The use of ultrasound to perform the fascia iliaca block was found to be superior when compared with the traditional approach using ‘loss-of-resistance’ to identify the correct plane, but still requires high volumes of local anesthetic20. At the level of a theoretical line drawn between the pubic tubercle and the anterior superior iliac spine, a linear high-frequency probe is used to identify the fascia iliaca and to guide the needle to the correct plane in transverse, short-axis view20.\n\n\nFemoral nerve block\n\nThe femoral nerve block represents the gold standard for analgesia of patients undergoing major knee surgeries21. With a high-frequency linear probe, the femoral nerve appears in a transverse section, as a triangular hyperechoic region, which lies lateral to the artery, deep to the fascia lata and to the fascia iliaca, and on the anterior aspect of the iliopsoas muscle22. Different authors have been evaluating whether the local anesthetic distribution and/or the catheter tip position may reduce the incidence of motor block maintaining the analgesic effect23,24. Ilfeld et al. showed that an anterior catheter tip positioning may increase cutaneous sensory block versus a posterior catheter tip placement, without increasing motor block23. Moreover, Szűcs et al. showed that depositing the local anesthetic only anteriorly to the nerve results in fewer needle redirections and greater patient satisfaction compared to surrounding the neural target circumferentially24. Farag et al. compared ultrasound guidance alone with either ultrasound guidance plus needle stimulation or ultrasound guidance plus catheter stimulation, in terms of postoperative pain, for insertion of femoral nerve catheters in more than 400 patients undergoing total knee arthroplasty25. The authors showed how ultrasound guidance alone represents the best approach to femoral perineural catheters25.\n\n\nSaphenous nerve block\n\nIn the last few years, the adductor canal block, a variant of the saphenous nerve block, has been presented as an alternative to the femoral nerve block in patients undergoing total knee replacement. In fact, the saphenous nerve block preserves quadriceps function in contrast to the femoral nerve block due to the fact that it is a pure sensory nerve26,27. A recent RCT has shown how the ultrasound-guided block of the saphenous nerve at the level of the adductor canal is superior to the block at the distal trans-sartorial level in terms of success rate, faster onset time, and better nerve visibility under ultrasound28. Nevertheless, it is important to recognize that the nerve to the vastus medialis also lies in the proximal portion of the adductor canal, possibly resulting in undesired motor weakness when a more proximal approach to the saphenous nerve is preferred. A cadaver study determined that in the majority of subjects (72.5%), the most distal point where it’s possible to see the nerve to the vastus medialis is where it transfixes the muscle proximal to the site where the saphenous nerve passes over the anterior surface of the superficial femoral artery to arrange medially to the vessel29. This anatomical knowledge is extremely important for deciding where to perform the block of the saphenous nerve, whether proximally or more distally.\n\n\nObturator nerve block\n\nSeveral approaches have been described to block the obturator nerve. The ultrasound-guided technique can be performed in-plane or out-of-plane and the nerve can be blocked before or after its bifurcation into the anterior and posterior branches. The patient is placed in a supine position and their thigh is externally rotated. The probe commonly used is a high-frequency linear probe, which is located perpendicular to the skin in an opposite position to angle between the inguinal crease and the adductor longus. This approach gives the possibility of correctly visualizing the structures of interest: the pectineus muscle, the adductor longus muscle, the adductor brevis muscle, and part of the adductor magnus muscle30,31. In this way, the operator can identify the anterior and the posterior divisions of the obturator nerve: the first one is located between the adductor longus and brevis muscles, while the second one is below the adductor brevis muscle30,31. Sinha et al. showed that an ultrasound-guided block in which 50% of the local anesthetic solution is injected between the pectineus muscle and the adductor brevis muscle and the rest of the anesthetic solution between the adductor brevis and the adductor magnus muscle allows one to obtain a mean reduction of muscular strength of 82% in almost all the patients evaluated (93%)32. Comparable results were achieved by Manassero et al. They conducted a RCT to determine whether interfascial spread of local anesthetic can supplant nerve stimulation as the end point for local anesthetic injection during ultrasound-guided obturator nerve block after the division of the obturator nerve33. The end point of their in-plane injection was correct interfascial spread of local anesthetic, defined as spread within the muscle interface, resulting in separation of target muscles. The authors demonstrated that this ultrasound-guided intrafascial injection is comparable with nerve stimulation33.\n\n\nParasacral sciatic nerve block\n\nIn the case of complex hip surgery, the gold standard is represented by the parasacral approach to the sciatic nerve due to the fact that with this proximal approach it is possible to block both the superior gluteal nerve and the nerve to the quadratus femoris muscle. Nevertheless, in comparison with more distal approaches to the sciatic nerve, the parasacral one is characterized by a potential increased risk of damage of the internal iliac vessels, ureter, rectum, and superior gluteal artery if the needle is advanced beyond the nerve2. Typically, the patient is placed in Sims’ position and a low-frequency curved probe is applied. In Taha’s approach, the small axis of the sciatic nerve is located at the posterior border of the ischium that usually can be identified as a curved hyperechoic line at this level34. The nerve lies deep to the piriformis muscle, lateral to the inferior gluteal vessels34. Whereas there is a high variability in the quality of image among patients, the nerve stimulator is considered useful to increase the reproducibility in this deep block.\n\n\nTrangluteal sciatic nerve block\n\nThe transgluteal approach is easier and safer compared with the parasacral one. At the transgluteal level, ultrasounds allow one to easily identify the greater trochanter, the ischial tuberosity, and the sciatic nerve, which is located between them35. At this level, if the physician inserts the needle beyond the nerve, he/she would commonly hit the bone and the only real risk is represented by the puncture of the inferior gluteal artery vessels. The patient assumes the Sims’ position and the operator uses a curved array low-frequency probe. The sciatic nerve is found beneath the gluteus maximus muscle halfway between the greater trochanter laterally and the ischial tuberosity medially35.\n\n\nSubgluteal sciatic nerve block\n\nAs previously reported, at the thigh level, the sciatic nerve can be recognized through the paraneural sheath that surrounds it. Two recent studies have analyzed the functional aspect of this space36,37. The first study, conducted on cadavers, showed that after injection of dye under the paraneurium, there was both distal and proximal spread along the nerve36. Instead, if the dye was injected outside the sheath, the spread was more much limited36. Ultrasound guidance allows an optimal image of the dissection36. The second study compared the injection of local anesthetic outside or under the paraneurium in patients undergoing subgluteal sciatic nerve block37. A three-dimensional reconstruction was made after the accomplishment of the block to quantify the spread of the local anesthetic solution along the nerve. Joining this technique with a clinical correlation about the quality of sensory block, it was possible for the authors to prove that an injection under the paraneurium allows better diffusion of local anesthetic and higher block efficacy than outside the paraneurium layer37. Karmakar et al.38 have definitively confirmed Andersen’s results36 and stated that the target for local anesthetic injection is the subparaneural space. To perform subgluteal or popliteal sciatic nerve blocks a linear high-frequency probe is usually sufficient. Although the patient can be in the ventral or supine position, the prone position is preferred. The ultrasound nerve localization can guide the choice of the puncture point in the posterior thigh. The local anesthetic should be placed all around the nerve between the epineurium and paraneurium. As reported by Krediet et al., the discrimination of the position of the needle tip is not always easy39. It is possible to identify the intraneural or extraneural needle tip position using an injection test of 0.5 mL, but even experts missed one of six intraneural injections39. A subparaneural injection not only accelerates the onset time but also increases the duration of the sensory blockade compared with circumferential extraneural injection40. If an extra-paraneurium injection is performed, the circumferential injection of local anesthetic around the nerve provides a higher success rate and shorter onset time than local anesthetic deposition at a single location next to the nerve41. On the contrary, when approaching the nerve at a subparaneural level, single injection or triple injections result in comparable success rates and total anesthesia related times41.\n\n\nAnkle block\n\nUltrasound-guided ankle block is frequently used and effective for foot surgeries. Compared with the conventional approach, the application of ultrasound increased the success rate and reduced the onset time of the ankle block42–44. To identify the four nerves of sciatic origin, a linear high-frequency probe is used. The tibial nerve is usually located between the medial malleolus and the Achilles tendon posterior to the posterior tibial artery and veins45. On the anterolateral aspect of the ankle next to the anterior tibial artery, the deep peroneal nerve can be identified. The superficial peroneal nerve is scanned before it becomes subcutaneous 10 to 15 cm proximal to the lateral malleolus. The sural nerve is located between the lateral malleolus and the Achilles tendon, close to the saphenous vein if it is visualizable45.\n\n\nConclusion\n\nIn conclusion, in the last few years, ultrasound guidance has allowed us to use different approaches targeted to the clinical context, optimizing differential block. A better comprehension of the sonoanatomy has suggested new perspectives in local anesthetic distribution around the target nerves, especially for the sciatic nerve, which is the most studied at the level of the popliteal fossa.\n\nDespite the heterogeneity of the studies analyzing the clinical impact of the ultrasound applied to the lower extremity blocks, current evidence shows a moderate improvement in performance time, patient procedural comfort, and in some cases even onset times compared to other localization techniques.", "appendix": "Competing interests\n\n\n\nThe authors disclose 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\nGhisi D, Delaunay L, Fanelli A: Use of ultrasound for lower extremity. Curr Opin Anaesthesiol. 2014; 27(5): 528–37. PubMed Abstract \| Publisher Full Text\n\nLiu SS: Evidence Basis for Ultrasound-Guided Block Characteristics Onset, Quality, and Duration. Reg Anesth Pain Med. 2015. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nSalinas FV: Ultrasound and review of evidence for lower extremity peripheral nerve blocks. Reg Anesth Pain Med. 2010; 35(2 Suppl): S16–25. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nLewis SR, Price A, Walker KJ, et al.: Ultrasound guidance for upper and lower limb blocks. Cochrane Database Syst Rev. 2015; 9: CD006459. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nLiu SS, Ngeow J, John RS: Evidence basis for ultrasound-guided block characteristics: onset, quality, and duration. Reg Anesth Pain Med. 2010; 35(2 Suppl): S26–35. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nMarhofer P, Greher M, Kapral S: Ultrasound guidance in regional anaesthesia. Br J Anaesth. 2005; 94(1): 7–17. PubMed Abstract \| Publisher Full Text\n\nPeripheral nerve blocks: a color atlas. Edited by Chelly JE - 3rd ed. Lippincott Williams & Wilkins, 2009. Reference Source\n\nBendtsen TF, Moriggl B, Chan V, et al.: Basic Topography of the Saphenous Nerve in the Femoral Triangle and the Adductor Canal. Reg Anesth Pain Med. 2015; 40(4): 391–2. PubMed Abstract \| Publisher Full Text\n\nMoayeri N, Groen GJ: Differences in quantitative architecture of sciatic nerve may explain differences in potential vulnerability to nerve injury, onset time, and minimum effective anesthetic volume. Anesthesiology. 2009; 111(5): 1128–34. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nKirchmair L, Entner T, Wissel J, et al.: A study of the paravertebral anatomy for ultrasound-guided posterior lumbar plexus block. Anesth Analg. 2001; 93(2): 477–81, 4th contents page. PubMed Abstract \| Publisher Full Text\n\nKirchmair L, Entner T, Kapral S, et al.: Ultrasound guidance for the psoas compartment block: an imaging study. Anesth Analg. 2002; 94(3): 706–10; table of contents. PubMed Abstract \| Publisher Full Text\n\nKarmakar MK, Ho AM, Li X, et al.: Ultrasound-guided lumbar plexus block through the acoustic window of the lumbar ultrasound trident. Br J Anaesth. 2008; 100(4): 533–7. PubMed Abstract \| Publisher Full Text\n\nKarmakar MK, Li JW, Kwok WH, et al.: Sonoanatomy relevant for lumbar plexus block in volunteers correlated with cross-sectional anatomic and magnetic resonance images. Reg Anesth Pain Med. 2013; 38(5): 391–7. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nKarmakar MK, Li X, Ho AM, et al.: Real-time ultrasound-guided paramedian epidural access: evaluation of a novel in-plane technique. Br J Anaesth. 2009; 102(6): 845–54. PubMed Abstract \| Publisher Full Text\n\nKarmakar MK, Li JW, Kwok WH, et al.: Ultrasound-guided lumbar plexus block using a transverse scan through the lumbar intertransverse space: a prospective case series. Reg Anesth Pain Med. 2015; 40(1): 75–81. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nBendtsen TF, Søballe K, Petersen EM, et al.: Ultrasound guided single injection lumbosacral plexus blockade for hip surgery anaesthesia. Brit J Anaesth. 2013. Reference Source\n\nBendtsen TF, Pedersen EM, Haroutounian S, et al.: The suprasacral parallel shift vs lumbar plexus blockade with ultrasound guidance in healthy volunteers--a randomised controlled trial. Anaesthesia. 2014; 69(11): 1227–40. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nDalens B, Tanguy A, Vanneuville G: Lumbar plexus blocks and lumbar plexus nerve blocks. Anesth Analg. 1989; 69(6): 852–4. PubMed Abstract \| Publisher Full Text\n\nRitcey B, Pageau P, Woo MY, et al.: Regional Nerve Blocks For Hip and Femoral Neck Fractures in the Emergency Department: A Systematic Review. CJEM. 2016; 18(1): 37–47. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nDolan J, Williams A, Murney E, et al.: Ultrasound guided fascia iliaca block: a comparison with the loss of resistance technique. Reg Anesth Pain Med. 2008; 33(6): 526–31. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nhttp://www.postoppain.org\n\nSzucs S, Morau D, Iohom G: Femoral nerve blockade. Med Ultrason. 2010; 12(2): 139–44. PubMed Abstract\n\nIlfeld BM, Loland VJ, Sandhu NS, et al.: Continuous femoral nerve blocks: the impact of catheter tip location relative to the femoral nerve (anterior versus posterior) on quadriceps weakness and cutaneous sensory block. Anesth Analg. 2012; 115(3): 721–7. PubMed Abstract \| Free Full Text \| F1000 Recommendation\n\nSzűcs S, Morau D, Sultan SF, et al.: A comparison of three techniques (local anesthetic deposited circumferential to vs. above vs. below the nerve) for ultrasound guided femoral nerve block. BMC Anesthesiol. 2014; 14: 6. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nFarag E, Atim A, Ghosh R, et al.: Comparison of three techniques for ultrasound-guided femoral nerve catheter insertion: a randomized, blinded trial. Anesthesiology. 2014; 121(2): 239–48. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nKim DH, Lin Y, Goytizolo EA, et al.: Adductor canal block versus femoral nerve block for total knee arthroplasty: a prospective, randomized, controlled trial. Anesthesiology. 2014; 120(3): 540–50. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nJæger P, Zaric D, Fomsgaard JS, et al.: Adductor canal block versus femoral nerve block for analgesia after total knee arthroplasty: a randomized, double-blind study. Reg Anesth Pain Med. 2013; 38(6): 526–32. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nMarian AA, Ranganath Y, Bayman EO, et al.: A Comparison of 2 Ultrasound-Guided Approaches to the Saphenous Nerve Block: Adductor Canal Versus Distal Transsartorial: A Prospective, Randomized, Blinded, Noninferiority Trial. Reg Anesth Pain Med. 2015; 40(5): 623–30. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nKapoor R, Adhikary SD, Siefring C, et al.: The saphenous nerve and its relationship to the nerve to the vastus medialis in and around the adductor canal: an anatomical study. Acta Anaesthesiol Scand. 2012; 56(3): 365–7. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nHelayel PE, da Conceição DB, Pavei P, et al.: Ultrasound-guided obturator nerve block: a preliminary report of a case series. Reg Anesth Pain Med. 2007; 32(3): 221–6. PubMed Abstract \| Publisher Full Text\n\nSimeoforidou M, Bareka M, Basdekis G, et al.: Peripheral nerve blockade as an exclusive approach to obturator nerve block in anterior cruciate ligament reconstructive surgery. Korean J Anesthesiol. 2013; 65(5): 410–7. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nSinha SK, Abrams JH, Houle TT, et al.: Ultrasound-guided obturator nerve block: an interfascial injection approach without nerve stimulation. Reg Anesth Pain Med. 2009; 34(3): 261–4. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nManassero A, Bossolasco M, Ugues S, et al.: Ultrasound-guided obturator nerve block: interfascial injection versus a neurostimulation-assisted technique. Reg Anesth Pain Med. 2012; 37(1): 67–71. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nTaha AM: A simple and successful sonographic technique to identify the sciatic nerve in the parasacral area. Can J Anaesth. 2012; 59(3): 263–7. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nChan VW, Nova H, Abbas S, et al.: Ultrasound examination and localization of the sciatic nerve: a volunteer study. Anesthesiology. 2006; 104(2): 309–14, discussion 5A. PubMed Abstract \| Publisher Full Text\n\nAndersen HL, Andersen SL, Tranum-Jensen J: Injection inside the paraneural sheath of the sciatic nerve: direct comparison among ultrasound imaging, macroscopic anatomy, and histologic analysis. Reg Anesth Pain Med. 2012; 37(4): 410–4. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nMissair A, Weisman RS, Suarez MR, et al.: A 3-dimensional ultrasound study of local anesthetic spread during lateral popliteal nerve block: what is the ideal end point for needle tip position? Reg Anesth Pain Med. 2012; 37(6): 627–32. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nKarmakar MK, Shariat AN, Pangthipampai P, et al.: High-definition ultrasound imaging defines the paraneural sheath and the fascial compartments surrounding the sciatic nerve at the popliteal fossa. Reg Anesth Pain Med. 2013; 38(5): 447–51. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nKrediet AC, Moayeri N, Bleys RL, et al.: Intraneural or extraneural: diagnostic accuracy of ultrasound assessment for localizing low-volume injection. Reg Anesth Pain Med. 2014; 39(5): 409–13. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nChoquet O, Noble GB, Abbal B, et al.: Subparaneural versus circumferential extraneural injection at the bifurcation level in ultrasound-guided popliteal sciatic nerve blocks: a prospective, randomized, double-blind study. Reg Anesth Pain Med. 2014; 39(4): 306–11. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nTiyaprasertkul W, Bernucci F, González AP, et al.: A Randomized Comparison Between Single- and Triple-Injection Subparaneural Popliteal Sciatic Nerve Block. Reg Anesth Pain Med. 2015; 40(4): 315–20. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nRedborg KE, Antonakakis JG, Beach ML, et al.: Ultrasound improves the success rate of a tibial nerve block at the ankle. Reg Anesth Pain Med. 2009; 34(3): 256–60. PubMed Abstract \| Publisher Full Text\n\nRedborg KE, Sites BD, Chinn CD, et al.: Ultrasound improves the success rate of a sural nerve block at the ankle. Reg Anesth Pain Med. 2009; 34(1): 24–8. PubMed Abstract \| Publisher Full Text\n\nChin KJ, Wong NW, Macfarlane AJ, et al.: Ultrasound-guided versus anatomic landmark-guided ankle blocks: a 6-year retrospective review. Reg Anesth Pain Med. 2011; 36(6): 611–8. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nLópez AM, Sala-Blanch X, Magaldi M, et al.: Ultrasound-guided ankle block for forefoot surgery: the contribution of the saphenous nerve. Reg Anesth Pain Med. 2012; 37(5): 554–7. PubMed Abstract \| Publisher Full Text" }	[ { "id": "12088", "date": "26 Jan 2016", "name": "Pierfrancesco Fusco", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12090", "date": "26 Jan 2016", "name": "Andrea Ortu", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-104
https://f1000research.com/articles/4-15/v1	16 Jan 15	{ "type": "Research Note", "title": "Evidence of polygenic selection on human stature inferred from spatial distribution of allele frequencies", "authors": [ "Davide Piffer" ], "abstract": "Spatial patterns of allele frequencies reveal a clear signal of natural (or sexual) selection on human height. The average frequency of 66 common genetic variants for 26 populations belonging to 5 sub-continental human groups was significantly correlated to average phenotypic population height. The method of correlated vectors provided additional evidence for a signal of natural selection in SNPs with higher significance. Factor analysis of the five top genome-wide association study (GWAS) hits revealed a clear factor indicating selection pressures on human height, peaking among northern Europeans and some African groups (Esan Nigeria) whilst reaching a nadir among South-East Asians.", "keywords": [ "Height", "Evolution", "Polygenic Selection", "Height" ], "content": "Introduction\n\nA recent GWAS (Wood et al., 2014) based on a very large sample (N=250K) identified common variants responsible for normal variation in human height within populations.\n\nOver the last few years, researchers have started moving away from the study of genetic evolution using a single-gene, Mendelian approach towards models that examine many genes together (polygenic). The more genes are involved in a given phenotype, the more the signal of natural selection will be “diluted” across different genomic regions (because each gene accounts for a tiny effect) making it difficult to detect it using approaches focused on a single gene (Pritchard et al., 2010; Piffer, 2014). A first attempt at empirically identifying polygenic selection was made by Turchin et al., (2012) on two populations (Northern and Southern Europeans) and evidence for higher frequency of height increasing alleles (obtained from GWAS studies) among Northern Europeans was provided. A drawback of that study was the reliance on populations from a single continent and that crude pairwise comparisons (e.g. French vs. Italian) were used without correlating frequency differences to average population height. Moreover, the strength of selection was not determined.\n\nTwo different approaches to identify selection based on the correlation of allele frequencies across different populations have been recently developed by Piffer (2013) and Berg & Coop (2014).\n\nPiffer’s method uses factor analysis of trait increasing alleles (found by GWA studies) as a tool for finding a factor that represent the strength of selection on a phenotype and the underlying genetic variation (Piffer, 2014a). An additional methodology consists of computing the correlation between genetic frequencies and the average phenotypes of different populations; then, the resulting correlation coefficients are correlated with the corresponding alleles’ genome-wide significance (p value). If the alleles contain selection signals, a positive correlation will be found, as alleles with high p value (more likely to be false positives) have a weaker correlation to average population phenotype (Piffer, 2014a).\n\nPiffer’s method (Piffer, 2013; Piffer, 2014a) to identify signals of polygenic selection was used in this study and applied to the top five GWAS hits (ranked according to p value). Piffer (2014b) carried out a study on height SNPs but it was based on a smaller GWAS sample and an older version (phase 1) of the 1000 Genomes data, containing data for only 14 populations. This paper uses the phase 3 1000 Genomes data and the GWAS meta-analysis was carried out on a much larger sample size, which produces more hits with better significance. The aim of this paper is to test the hypothesis that stature has undergone natural or sexual selection in populations after humans dispersed in different continents giving rise to distinct genetic clusters.\n\n\nMethods\n\nFrequencies of alleles with a positive effect (height increasing) were obtained from 1000 Genomes (phase 3): http://browser.1000genomes.org/index.html comprising 26 populations belonging to five racial groups.\n\nAverage population height was obtained from the references listed at: http://en.wikipedia.org/wiki/Human_height, considering only statistics published after 2000 and young age groups (18–40). Only 11 populations met these criteria (see references in Table 1).\n\n*Not on Wikipedia. Region of a country, more specific statistics found elsewhere.\n\nFor each chromosome, the three alleles with the highest p values were selected, and these were all unlinked (>500Kb apart from each other). Only unlinked alleles were used to avoid the confounding influence of linkage on cross-population allele frequency. Selection was restricted only to the alleles with the highest significance because these are less likely to be false positives. The same number of SNPs (3) from each chromosome was used to get a representative sample of the entire genome, to avoid bias due to chromosome location. The conventional nominal p-value < 5×10-8 was used as significance threshold (Barsh et al., 2012).\n\nA polygenic score was calculated as the mean frequency of height increasing alleles (defined as those with a positive Beta coefficient in the meta-analysis).\n\nAnalyses were carried out using R.\n\n\nResults\n\nPolygenic scores and average country height are reported in Table 1. The Pearson correlation between polygenic score and average country height was r=0.83 (N=11, p=0.002). Table 2 reports average frequencies by sub-continental populations.\n\nFrequencies in descending order are: 1) Africans (AFR); 2) Europeans (EUR); 3) South Asians (SAS); 4) Latin Americans/Hispanics (AMR); 5) East Asians (ASN).\n\nSpearman’s rank order correlation between each allele’s p value and its correlation with the polygenic score and with height were respectively -0.26 and -0.34 (N=66, p=0.037 and 0.0053). The “rcorr” and “cor” functions in R produced slightly different results due to differences in dealing with ties (equal values). “cor” produced slightly stronger coefficients (-0.28 and -0.37).\n\nThis provides evidence for the hypothesis that more significant GWAS hits (alleles) are enriched with natural selection signal. A similar phenomenon was observed in a previous analysis of genes affecting human height (Piffer, 2014b).\n\nFactor analysis requires a satisfying cases to variable ratio, thus only a handful of SNPs could be used and these had necessarily to be those with the lowest p value, as they are more likely to be genuine hits (see previous section, MCV).\n\nThe top 5 alleles (i.e. those with the lowest p value) all correlated with the polygenic score and with average height in the expected direction (positively), as shown in Table 3 (see Dataset 2).The average correlations were 0.58 and 0.69, respectively, which is a significant improvement compared to the average of the correlations with polygenic score and height of all the 66 alleles (r=0.03 and 0.04, respectively; see Dataset 1, cells BP38–39).\n\n(p value and r with polygenic (pol) score).\n\nA factor analysis using minimum residuals was carried out. A single factor was extracted that explained 42% of the variance. Factor loadings are displayed in Table 4. These are all positive (in the expected direction).\n\nStandardized loadings (pattern matrix) based upon correlation matrix.\n\nFactor scores were extracted with the Thurstone method (Thurstone, 1947), and are reported in Table 5.\n\nThe Pearson correlation between average country height and the factor score was strongly positive (r=0.88, N=11, p=0.001). This factor was also significantly correlated to the polygenic score (r=0.78, N=26, p<0.001).\n\n\nDiscussion\n\nA polygenic score, created by averaging frequencies from 26 populations of 66 height increasing alleles by the largest and most recent human height GWAS, was positively correlated with the average height of 11 populations. The method of correlated vectors revealed that alleles with lower p values had a higher correlation with phenotypic height and polygenic score, suggesting that they tend to be enriched with signal of natural selection. A factor analysis of the top five GWAS hits produced a factor (whose loadings are all in the expected direction) which is significantly and strongly correlated both to population average height and to polygenic score. This showed an improvement over the correlation of the five single alleles with population height (Table 3, last row) which averaged 0.66, which in turn improved over the average correlation of the 66 alleles, which was near zero.\n\nThe rankings of polygenic scores match with the folk perception on the stature of various racial groups: Africans> Europeans> South/Central Asians> Hispanics> East Asians (Table 2).\n\nSouth East Asians had the lowest scores, a result which matches with their anthropometric description.\n\nWithin Europe, northern Europeans (Finns and White Americans) had a higher genotypic stature than their southern counterparts (Italians and Spaniards), confirming the results from a previous study on GWAS loci which compared northern vs southern Europeans (Turchin et al., 2010).\n\nA limitation was the unavailability of sound statistics on the average height of many populations. Moreover, although human height is largely heritable, it is also heavily influenced by nutrition and living conditions. The importance of environment is suggested by the dramatic secular trend which took place in the 20th century in developed countries (e.g. Arcaleni, 2006; Webb et al., 2008); an association with dietary intakes (i.e. milk consumption) and socioeconomic status has also been observed (Mamidi et al., 2011; Webb et al., 2008). Most of the missing data were for developing countries which likely have not reached their full growth potential or ethnic groups living in Western societies (Indian Telegu or Gujarati) for which anthropometric statistics are not easily available. If the allele frequency factor represents a genuine signal of natural selection, then the difference between it and current phenotypic height could be used as an indicator of the quality of diet and living conditions in general.\n\n\nConclusion\n\nFactor analysis of allele frequencies is a promising method for detecting signals of recent selection on polygenic traits.\n\n\nData availability\n\nF1000Research: Dataset 1. Hits 1+2+3. 10.5256/f1000research.6002.d41833 (Piffer, 2014c).\n\nF1000Research: Dataset 2. Method of correlated vectors (MCV). 10.5256/f1000research.6002.d41834 (Piffer, 2014d).", "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\nArcaleni E: Secular trend and regional differences in the stature of Italians, 1854–1980. Econ Hum Biol. 2006; 4(1): 24–38. PubMed Abstract \| Publisher Full Text\n\nBarsh GS, Copenhaver GP, Gibson G, et al.: Guidelines for genome-wide association studies. PLoS Genet. 2012; 8(7): e1002812. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBerg JJ, Coop G: A population genetic signal of polygenic adaptation. PLoS Genet. 2014; 10(8): e1004412. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCacciari E, Milani S, Balsamo A, et al.: Italian cross-sectional growth charts for height, weight and BMI (6–20 y). Eur J Clin Nutr. 2002; 56(2): 171–80. PubMed Abstract\n\nHung MV, Pak S: The impact of environment on morphological and physical indexes of Vietnamese and South Korean students. VNU Journal of Science, Natural Science and Technology. 2008; 24: 50–55. Reference Source\n\nMamidi RS, Kulkarni B, Singh A: Secular trends in height in different states of India in relation to socioeconomic characteristics and dietary intakes. Food Nutr Bull. 2011; 32(1): 23–34. PubMed Abstract\n\nMcDowell MA, Fryar CD, Hirsch R, et al.: “Anthropometric Reference Data for Children and Adults: United States, 2003–2006”. National Health Statistics Reports. 2008; 10. Reference Source\n\nMeisel A, Vega M: “A tropical success story: a century of improvements in the biological standard of living, Colombia 1910–2002”. Paper prepared for The Fifth World Congress of Cliometrics, Venice International University, Venice, Italy, July 8–11, 2004. Reference Source\n\nMinistry of Education, Culture, Sports, Science and Technology. Japan, 2004. Reference Source\n\nMoody A: 10: Adult anthropometric measures, overweight and obesity. In Craig, Rachel; Mindell, Jennifer. Health Survey for England – 2012. 2013. Reference Source\n\nNational Institute for Health and Welfare: Lasten kasvunseurannan uudistaminen, Asiantuntijaryhmän raportti. 2011. Reference Source\n\nPiffer D: Factor Analysis of Population Allele Frequencies as a Simple, Novel Method of Detecting Signals of Recent Polygenic Selection: The Example of Educational Attainment and IQ. Mankind Quarterly. 2013; 54(2): 168200. Reference Source\n\nPiffer D: Simple statistical tools to detect signals of recent polygenic selection. IBC. 2014a; 6(1); 1–6. Reference Source\n\nPiffer D: Opposite selection pressure on stature and intelligence across human populations. Open Behavioral Genetics. 2014b. Reference Source\n\nPiffer D: Hits 1+2+3. F1000Research. 2014c. Data Source\n\nPiffer D: Method of correlated vectors (MCV). F1000Research. 2014d. Data Source\n\nThurstone LL: Multiple Factor Analysis. University of Chicago Press. 1947. Publisher Full Text\n\nTurchin MC, Chiang CW, Palmer CD, et al.: Evidence of widespread selection on standing variation in Europe at height-associated SNPs. Nat Genet. 2012; 44(9): 1015–1019. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWebb EA, Kuh D, Pajak A, et al.: Estimation of secular trends in adult height, and childhood socioeconomic circumstances in three Eastern European populations. Econ Hum Biol. 2008; 6(2): 228–236. PubMed Abstract \| Publisher Full Text\n\nWood AR, Esko T, Yang J, et al.: Defining the role of common variation in the genomic and biological architecture of adult human height. Nat Genet. 2014; 46(11): 1173–86. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYang XG, Li YP, Ma GS, et al.: [Study on weight and height of the Chinese people and the differences between 1992 and 2002]. Zhonghua Liu Xing Bing Xue Za Zhi. 2005; 26(7): 489–93. PubMed Abstract" }	[ { "id": "9015", "date": "30 Oct 2015", "name": "Ben Busby", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe only major thing that, in my opinion, stands between this manuscript is the availability of the R scripts used to produce the data tables (especially given the difference between corr and rcorr). I looked for any linking to or availability of these scripts in in Piffer 2013 or 2014a-d, and could not find it. Without these scripts, I do not think this work can be considered reproducible. Two additional (minor) revisions are as follows.First, the phenotypic data in table one seems to reply solely on Wikipedia. It seems likely that the author could find additional height information beyond Wikipedia.Second, the last four paragraphs of the article could be combined into one.", "responses": [] } ]	1	https://f1000research.com/articles/4-15
https://f1000research.com/articles/5-103/v1	25 Jan 16	{ "type": "Review", "title": "Renal Replacement Therapy", "authors": [ "Zaccaria Ricci", "Stefano Romagnoli", "Claudio Ronco", "Stefano Romagnoli", "Claudio Ronco" ], "abstract": "During the last few years, due to medical and surgical evolution, patients with increasingly severe diseases causing multiorgan dysfunction are frequently admitted to intensive care units. Therapeutic options, when organ failure occurs, are frequently nonspecific and mostly directed towards supporting vital function. In these scenarios, the kidneys are almost always involved and, therefore, renal replacement therapies have become a common routine practice in critically ill patients with acute kidney injury. Recent technological improvement has led to the production of safe, versatile and efficient dialysis machines. In addition, emerging evidence may allow better individualization of treatment with tailored prescription depending on the patients’ clinical picture (e.g. sepsis, fluid overload, pediatric). The aim of the present review is to give a general overview of current practice in renal replacement therapies for critically ill patients. The main clinical aspects, including dose prescription, modality of dialysis delivery, anticoagulation strategies and timing will be addressed. In addition, some technical issues on physical principles governing blood purification, filters characteristics, and vascular access, will be covered. Finally, a section on current standard nomenclature of renal replacement therapy is devoted to clarify the “Tower of Babel” of critical care nephrology.", "keywords": [ "Renal Replacement Therapy", "acute kidney injury", "dialysis", "anticoagulation strategies", "blood purification", "critical care nephrology" ], "content": "Introduction\n\nCritically ill patients with severe diseases and multisystem organ failure are currently frequently admitted to and treated in the intensive care unit (ICU). Although the identification and management of multisystem organ failure has improved, the incidence has increased over the last half-century1. Therapeutic options in the setting of multisystem organ failure are mostly aimed at supporting vital functions. The kidneys are almost always involved in such a syndrome, and dialytic techniques are routinely used in the ICUs to treat severe acute kidney injury (AKI)2.\n\nCurrent practice in renal replacement therapy (RRT) for adult critically ill patients, with specific details on technical features and clinical applications, will be reviewed.\n\n\nTechnical issues\n\nPeter Kramer in 1977 described the first continuous form of dialysis specifically dedicated to critically ill patients: continuous arterio-venous hemofiltration (CAVH)3. In CAVH, blood flow in the circuit was driven by a spontaneous arterio-venous pressure gradient and spontaneous ultrafiltration (UF) occurred depending on the transmembrane pressure (TMP) gradient. The arterio-venous pressure gradient was dependent on the mean arterial pressure of the patient and the intrinsic resistance of the circuit (determining the blood flow); the UF was determined by the hydrostatic pressure drop inside the filter and the negative suction provided by the UF column from the patient level to the ground. As a consequence, patients with low blood pressure and/or low cardiac output achieved the lowest clearances but were able to self-limit UF. When peristaltic pumps were added to the extracorporeal circuit, veno-venous hemofiltration became feasible. Subsequently, fluid delivery systems and UF control mechanisms were implemented allowing dialysate and replacement solutions to be delivered with acceptable accuracy. Higher clearances were finally possible because of the ability to provide increased flow rates. Although clearly improved, RRT machines were still inaccurate, and safety and performance were still a challenge. It soon became evident that an ideal extracorporeal circuit requires continuous pressure measurements at different levels (inlet and outlet of vascular access, inlet and outlet of the filter and UF ports)4.\n\nCurrently, third/fourth-generation machines are designed to meet the dialysis dose requirements and the strict safety features that are recommended in every modern ICU5. Contemporary devices, equipped with 4 to 5 roller pumps, 3 to 4 scales and pressure sensors, allow a fluid load from 20 to 40 kg in order to reduce nursing workload. In addition, maximal flow rates have increased up to about 450 mL⁄min for the blood pump, 8–10 L⁄hr for the dialysate⁄replacement pumps, and 20–25 L⁄hour for the effluent pump. Mechanical implementation has been associated with a huge electronic evolution: interfaces have been implemented with wide screens and clear alarm signals and warnings, and circuit pressure trends are now visualized. Furthermore, during continuous RRT (CRRT), now flexible and safe, modalities can be switched in order to tailor them to the patients’ need. Modern RRT filters, a key component of the system, are composed of groups of hollow fibers with a range of surface areas (from 0.1 to over 2 m2) in order to meet the need of differently sized patients. Such fibers have a generally high porosity (30–50 A°) with a pore cutoff size of 30 kDa and are used for both diffusive and convective treatments. Polyacrylonitrile, polysulphone and poly(methyl methacrylate) (PMMA) are the most commonly used membranes and allow a high UF coefficient (over 20 ml/h/mmHg) and high diffusive and convective performance. Biocompatibility (the change in blood factors induced by membrane/blood contact) is considered the most important quality of these RRT membranes.\n\nRenal replacement consists of blood purification by semi-permeable membranes. Blood flows into hollow fibers composed of porous biocompatible synthetic material. A wide range of substances (water, urea, low, middle and high molecular weight solutes) can be transported across such membranes, from the blood to the effluent side of the hollow fibers, by the mechanism of diffusion (solutes) and convection (water and solutes) (Figure 1).\n\nDuring diffusion solutes flux (Jx) is a function of: solutes concentration gradient (dc) between the two sides of the semi-permeable membrane, temperature (T), diffusivity coefficient (D), membrane thickness (dx) and surface area (A) according to the following equation: Jx = D T A (dc/dx)\n\nConvective flux of solutes (Jf) requires instead a pressure gradient between the two sides of the membrane (transmembrane pressure TMP), that moves a fluid (plasma water) with its « crystalloid » content (a process called ultrafiltration, whose entity is also dependent on membrane permeability coefficient (Kf). Colloids and cells will not cross the semipermeable membrane, depending on the pores’ size. Jf = Kf × TMP\n\nDialysis is based on the diffusion principle: a dialytic solution flows through the filter counter current to blood flow in order to maintain the highest solute gradient from inlet to outlet port. Diffusion is the solute transport method applied during intermittent hemodialysis (IHD) and continuous veno-venous hemodialysis (CVVHD) (Figure 2). During diffusion, the movement of solutes depends on their tendency to reach the same concentration on each side of the membrane, allowing the passage of solutes from the compartment with the highest concentration to the compartment with the lowest concentration. Other components of the semi-permeable membrane that affect diffusion include thickness and surface area, dialysate temperature, and diffusion coefficient.\n\nBlack triangle represents blood flow direction; gray triangle indicates dialysate/replacement solutions flows. V-V: veno-venous; Uf: ultrafiltration; Rpre: replacement solution prefilter; Rpost: replacement solution postfilter; Do: dialysate out; Di: dialysate in; Qb: blood flow; Quf: ultrafiltration flow; Qf: replacement solution flow; Qd: dialysate solution flow.\n\nDuring convection, solutes are transported across a semi-permeable membrane by UF (water transfer across the membrane). In other words, as the solvent (plasma water) is pushed (ultrafiltered) across the membrane according to the TMP, solutes are carried with it, as long as the porosity of the membrane allows the molecules to be sieved from the blood. Convection is applied during continuous veno-venous hemofiltration (CVVH) while the combination of both convection and diffusion configures continuous veno-venous hemodiafiltration (CVVHDF) (Figure 2).\n\nThe UF rate (QUF) in CAVH systems was governed by the membrane UF coefficient (Km) and the TMP gradient generated by the pressures on both sides of the hollow fiber according to the following formula:\n\nQUF = Km * TMP\n\nIn modern RRT machines QUF is regulated by a pump and, consequently, it is constantly maintained regardless of whether the filter is “fresh” (when UF occurs with low TMP) or clogging (in which case a progressive secondary increase of TMP is observed). In fact, as molecules cleared during convection are physically dragged to the UF side, the protein layering that progressively clogs the fiber pores significantly limits solute transport6. A peculiar membrane capacity, defined as adsorption, has been shown to play a major role in higher molecular weight toxins7; however, membrane adsorptive capacity is generally saturated within the first few treatment hours. This observation explains the minimal impact of the adsorption component on solute clearance8. An exception on this rule is made by high-mobility group box 1 protein (HMGB-1), as this major sepsis key mediator can be significantly removed (more than 90%) by adsorption through an acrylonitrile-treated surface (AN69-ST) and PMMA membranes9. However the clinical relevance of this molecule clearance remains to be ascertained. As UF proceeds and plasma water and solutes are filtered from blood, hydrostatic pressure within the filter declines and the effect of oncotic pressure increases because blood concentrates and hematocrit increases. The fraction of plasma water that is removed from blood during UF is called the filtration fraction and should be kept in the range of 20–25% in order to avoid equalization of the oncotic pressure to the TMP and filtration/pressure equilibrium. Finally, replacing the plasma water removed through the filter with a substitution solution completes the hemofiltration process and purified blood is returned to the patient. When the substitution fluid is administered after the filter it is referred to as post-dilution HF. When the substitution solution is infused before the filter it is referred to as pre-dilution HF. While post-dilution allows a urea clearance equivalent to therapy delivery (see below), pre-dilution, in spite of theoretical reduced solutes clearances, allows prolonged circuit lifespan by reducing hemoconcentration and protein caking effects within filter fibers. The difference between the volume of ultrafiltered plasma water and reinfused substitution solution gives the net UF, which is the fluid that is eventually removed from the patient for fluid control. Net UF prescription is based on patient needs and can range from more than 1 L/h (pulmonary edema in a patient with congestive heart failure and diuretic-resistant AKI) to zero (sepsis with catabolic state increased creatinine levels and conserved diuresis). A net UF rate must be added to diffusion-based CRRT modalities in order to achieve fluid balance control since diffusion does not allow for water exchanges.\n\nInterestingly, apart from the demonstration of different clearances of middle molecular weight solutes (i.e beta-2 microglobulin) provided by CVVH when compared to similar CVVHD doses8, no study so far has shown that the application of hemofiltration, with respect to hemodialysis, improves hard outcomes (such as mortality, length of mechanical ventilation, length of hospital stay)10,11.\n\nRRT dose is a measure of the quantity of blood purified by “waste products and toxins” and is generally expressed as clearance (K). Clearance is defined as the amount of blood purified by a single solute in the unit of time and it is expressed as volume over time, as it represents the flow of “cleaned” blood. As these still incompletely known substances “to be purified” are difficult to measure and quantify, the operative view of RRT dose is generally reduced to the measure of the elimination of a representative marker solute. Unfortunately, the marker solute does not represent all the solutes that accumulate during AKI because kinetics and volume of distribution are different for each solute and its removal during RRT is not necessarily representative of the removal of other solutes. However, since single solute marker assessment of dialysis dose appears to be related to patient outcome12, urea and creatinine, due to their significant accumulation during AKI and the ease of their routine daily blood determination, are generally used as reference solutes for measuring renal replacement clearance during either chronic or acute dialysis.\n\nDuring RRT, clearance depends upon blood flow rate (Qb), substitution flow rate (Qf) or dialysis flow (Qd), solute molecular weights, and hemodialyzer type and size. Qb is mainly dependent upon vascular access and the operational characteristics of utilized machines in the clinical setting. Qf is strictly linked to Qb, during convective techniques, by filtration fraction. Filtration fraction does not limit Qd, but when Qd/Qb ratio exceeds 0.3, dialysate will not be completely saturated with blood diffusing solutes. When UF is applied, molecules are dragged with plasma water through the filter pores according to their sieving coefficient (SC); the SC is calculated as the effluent/plasma concentration ratio of the target molecule. When the SC is 1, as in the case of small molecules (below 12 kD, such as creatinine and urea), the same solute concentration is found in the two sides of the hollow fiber. A SC value of 0 means that the molecule is not filtered (i.e. albumin, hemoglobin, etc). K during convection is measured by the product of Qf multiplied by the SC; hence, there is a linear relationship between K and Qf, the SC being the changing variable for different solutes. During diffusion, the linear relationship is lost when Qd exceeds about 1/3 of Qb.\n\nOne of the crucial merits of specific dose prescription, calculation, and delivery is the avoidance of underdialysis and the improved monitoring and awareness of effective delivered therapy.\n\nBlood purification can be achieved in the ICU both by continuous and intermittent RRT. In theory, during continuous RRT, the treatment is kept running 24 hours a day, seven days a week. During intermittent RRT renal support is delivered in intermittent sessions lasting (depending on center preferences, protocols, and the patient’s clinical status) 3–6 hours, typically three times per week (or depending on specific needs). Currently, about 80% of critically ill patients are treated with continuous RRT. However, due to the absence of significant differences in outcome deriving from the application of continuous vs intermittent RRT, no specific recommendation is provided by the major critical care societies, and the choice is mainly left to institutional protocols and expertise. Apart from evidence in large clinical trials, more gentle RRT application is generally better tolerated in hemodynamically unstable, critically ill patients with severe AKI. Furthermore, since the occurrence of intradialytic hypotension is proportional to the net Quf rate, it is possible to prescribe a lower net Quf rate when the treatment is applied over 24 hours as compared to a quick 3-hour session. Recently, the Surviving Sepsis Campaign guidelines for management of severe sepsis and septic shock13 concluded that, based on present scientific evidence, continuous RRT should be considered equivalent to IHD for treatment of AKI. Vinsonneau and colleagues14 conducted a large, prospective, randomized multicenter study in 21 ICUs over a 3.5-year period. The primary end point was the 60-day mortality following the randomization of 360 patients with AKI to either CVVHDF or IHD and no difference was found in 28, 60 and 90-day mortality between the two groups. Hence, according to the results, the study investigators concluded that IHD can be delivered safely to critically ill patients. Unfortunately, delivered dose in both arms was not controlled for in the trial design. The accompanying editorial15 emphasized that the advantages of continuous therapies are particularly significant when therapy downtime is minimized, in order to enhance the low intensity, smooth and continuous effects of plasma K. However, Vinsonneau et al.’s findings have been confirmed repeatedly by other studies16,17. One of the reasons for the lack of hard outcome differences between intermittent and continuous techniques could be because IHD has become safer and more efficacious18. Alternatively, a liberal application of CRRT (including extended and probably wrong indications) can cause adverse effects as discussed later below.\n\nHybrid techniques, which combine the advantages of both continuous and intermittent modalities, may represent an interesting compromise. Although a variety of names have been given to hybrid techniques (see the nomenclature section)19–22 depending on variations in schedule and type of solute removal (convective or diffusive), they all attempt to provide a gentle, prolonged and more feasible extended IHD, with all the advantages of discontinuous treatment (less need for anticoagulation, increased patient mobility, easier possibility of fitting prescribed schedules without downtime). These techniques generally have shown good results in terms of hemodynamic tolerance and adequacy of dialytic dosage23. Baldwin and coworkers compared 3 consecutive days of CVVH with a similar period of extended daily dialysis with filtration23. No significant difference was found between the two therapies as far as urea or creatinine levels and electrolyte and acid–base control. Interestingly, after 3 days of treatment, there was a mild but persistent metabolic acidosis in the extended dialysis group, but in the CVVH group hypophosphatemia was described. Advantages and disadvantages of IHD, CRRT and hybrid techniques, respectively, are depicted in Table 1.\n\nIn conclusion, intermittent and continuous therapies, when applied by expert centers, may appear similar where hard outcomes are concerned. As far as long-term RRT outcomes are concerned, however, recent reports indicate that RRT survivors treated by IHD might have a lower chance of recovering pre-morbid kidney function and have an increased risk of remaining dialysis-dependent at hospital discharge24.\n\nThe contact between blood and artificial surfaces induces activation of the coagulation cascade, resulting in filter and/or circuit clotting and the need for anticoagulation25–29. Anticoagulation strategy depends on the type of RRT and is often needed for continuous therapies due to the increased exposure to the blood-artificial surface. Aims of anticoagulation are: maintenance of extracorporeal circuit and dialyzer patency; reduction of downtime that might have a clinical impact in the overall RRT clearance; reduction of treatment cost by the utilization of less material; and achievement of the above aims while minimizing risks for the patient. Several technical features of the RRT circuit are likely to affect the success of any anticoagulant approach: vascular access has to be of adequate size; tube kinking should be avoided; blood flow rate should exceed 100 ml/min; pump flow fluctuations must be prevented; and the venous bubble trap, where air/blood contact occurs, must be accurately monitored. Furthermore, plasma filtration fraction should be kept as far as possible below 20% and, when possible, pre-dilution hemofiltration should be selected. There is evidence that, when circuit set-up is perfectly optimized, anticoagulants are only a relatively minor component of circuit patency. When patients have altered coagulation, thrombocytopenia, or active bleeding (e.g. after trauma or surgery), RRT can be safely performed without anticoagulation25. Lastly, regional citrate anticoagulation (RCA) can be safely used nowadays not only in patients with bleeding risks but also in patients without bleeding risks, according to the current KDIGO guidelines30. Extensive training is needed regarding the metabolic side effects of citrate before embarking upon routine citrate anticoagulation. In recent years, new commercially available citrate solutions together with adapted CRRT machines have rendered the technique safer and easier to use31.\n\nDifferent methods for anticoagulation are summarized in Table 2.\n\nAbbreviations: RRT, renal replacement therapy; UHF, unfractioned heparin; HIT, heparin-induced thrombocytopenia; aPTT, activated prothrombin time; LMWH, low molecular weight heparin; PGI2, prostacyclin.\n\n\nClinical applications\n\nRegardless of RRT technique used, the following clinical variables are typically compromised in the critically ill patient with AKI: fluid status and tissue edema, hemodynamics, acid–base and electrolyte equilibrium, protein-rich nutritional support, phosphate and calcium balance, and infection control.\n\nCurrently, a broader concept of “timely intervention” is generally accepted. When oliguria results in impairment of one or more of the above clinical variables, RRT should be instituted rapidly in order to avoid fluid overload and congestion. The only urgent indications to perform dialysis are pulmonary edema refractory to high dose diuretics, rapidly increasing hyperkalemia, severe refractory acidosis, symptoms/signs of uremia, and specific drug intoxications. Critically ill patients, especially if they are oliguric or anuric, typically gain weight from water accumulation and large volumes of intravenous fluids. In such patients, water removal is indicated for the achievement of a negative daily fluid balance, which has been associated with multiorgan function improvement (i.e. at the pulmonary, cardiac and renal level) in observational and retrospective studies32,33. Furthermore, a slow continuous RRT with fluid removal over 24 hours is better able to manage the critically ill patient’s needs; in case of increased nutritional administration, fluids deriving from parenteral drugs or hemoderivates transfusion, QUF can be easily tailored on an hourly base34. Conversely, rapid (or, worse, intermittent) ultrafiltration of body water may lead to acute hypovolemia and subsequent hypotension, since refilling from the interstitial compartment is slow and steady due to hydrostatic and osmotic pressures35. Since no effective clinical monitoring is currently available to “measure” fluid overload and the amount of fluid excess to be removed, clinical expertise in critical care nephrology (and possibly a multidisciplinary approach) is essential for adequate management of fluid removal36.\n\nAs for the exact timing for starting RRT, a definition of timing is currently not available. Timing can be considered as a synonym for “indication” and then one can start CRRT early or late depending on how severe (or conventional) the indication is (e.g. creatinine level or potassium level or the presence of sepsis)37. Otherwise, timing can be considered as the time elapsed between any established indication to start and the effective inception of the dialytic session. A recent retrospective study38 confirmed that crude 90-day mortality of patients with RRT started after “classic indications” (identified as hyperkalemia, severe acidosis, urea above 100 mg/dl, oliguria or anuria and fluid overload with pulmonary edema) was significantly higher than in patients with “pre-emptive” RRT (initiated without any conventional indication): adjusted odds ratio, 2.05; 95% CI, 1.03 to 4.09. Interestingly, also patients with classic RRT but a delayed start (>12 hours from indication) showed higher crude mortality compared with patients with classic RRT that started early due to urgent indications (<12 hours from indication); this association persisted after adjustment for known confounders (odds ratio, 3.85; 95% CI, 1.48 to 10.22). Due to the retrospective nature of this study, it is not clear how effectively comparable the two populations (classic vs pre-emptive or early vs delayed) are. The Canadian Critical Care Trials Group recently concluded the first pilot trial aiming to prospectively evaluate the feasibility of a protocol-driven accelerated RRT initiation39. This interesting complex trial showed how difficult it would be to conduct a large multicenter prospective trial attempting to randomize two AKI populations only differing by the time elapsed from RRT indication to treatment start. In fact, in the pilot trial, a large number of patients had to be excluded after provisional eligibility per protocol design: those deemed by the intensivist and nephrologist in charge as requiring urgent RRT or deferral of RRT indication. This excluded cohort might unfortunately represent a sample of patients whose outcomes are potentially affected by pre-emptive or delayed RRT start. However, in the analysis of the enrolled 101 patients, the authors succeeded in their primary outcome. In the accelerated arm, median time to RRT start was 7.4h. In the standard arm, 33 patients started RRT at a median of 31.6h from eligibility, and, interestingly, the other 19 did not receive any RRT (6 died and 13 recovered kidney function). Even though these preliminary results should be interpreted with caution, hard outcomes were not affected by acceleration of RRT start (mortality was 38% in the accelerated and 37% in the standard arm).\n\nFrom a clinical standpoint, the effects of RRT dose have been systematically evaluated in the last 10 years. After the milestone trial from the group in Vicenza back in 200040, CVVH dose has been indexed for the first time to patients’ body weight (mL/Kg/h), in order to highlight that this variable is of high importance in AKI patients. Two large, multicenter, randomized controlled studies published in 2009 (the randomized evaluation of normal versus augmented level (RENAL) replacement therapy study41 and in 2008, the VA/NIH Acute Renal Failure Trial Network (ATN) study42) finally clarified the concept of optimal dialysis dose. These fundamental trials were conceived to test the hypothesis concerning the impact of “intensive” RRT on hard outcomes (namely mortality and ICU stay) when compared to “less intensive” renal support. The RENAL study was conducted exclusively with continuous therapies (as this is the standard in Australia) and compared 25 mL/Kg/h CVVHDF to 40 mL/Kg/h. Using a different approach, the ATN study, conducted in North America, considered 20 mL/Kg/h CVVHDF or thrice weekly intermittent dialysis as the control group and compared it to 35 mL/Kg/h CVVHDF or daily IHD as the intensive arm. Apart from methodological differences, both studies confirmed that “intensive” RRT does not improve patient outcomes, and survival (although different between Australia’s and United States’ centers) was similar among compared arms. Based on the results of those trials, the accepted dose of RRT is considered to be within the range of 25–35 mL/Kg/h for CRRT and/or thrice weekly IHD with a Kt/V (see table on nomenclature) of 1.3.\n\nClearly, clinical effects of RRT dose are not limited to urea and excess body water control. Oligo anuric patients often suffer from mild acidemia secondary to increased unmeasured anions (strong ion gap – SIG - 12.3 mEq/l), hyperphosphatemia, and hyperlactatemia. This acidosis is attenuated by the alkalizing effect of hypoalbuminemia. The effect on acid–base balance of IHD and CVVHDF has been evaluated43: metabolic acidosis is common in both groups and both techniques correct metabolic acidosis; however, the rate and degree of correction may significantly differ between continuous and intermittent techniques. In the same study43 CVVHDF was shown to normalize metabolic acidosis more rapidly and more effectively during the first 24 hours than IHD. IHD was also associated with a higher incidence of metabolic acidosis as compared to CVVHDF during the subsequent 2-week treatment period. Accordingly, continuous RRT could be considered physiologically superior to IHD in the correction of metabolic acidosis. In a comparison between CVVH and peritoneal dialysis, all patients receiving CVVH achieved correction of acidosis by 50 hours of treatment, whereas only 15% of those randomized to peritoneal dialysis achieved such correction (P < 0.001)44. Despite the results of these studies, correction of acidosis by RRT has not revealed any specific impact on outcomes.\n\nAlthough safety features of CRRT machines have evolved, the possibility that CRRT may confer increased risk should not be overlooked45. In fact, as with any type of continuous extra corporeal therapy, CRRT often requires continuous anticoagulation therapy, which can increase the bleeding risk in case of heparin use or metabolic derangements in case of citrate use. Conversely, clotting of the extracorporeal circuit also occurs frequently with CRRT, which might contribute to blood loss and could exacerbate anemia in critically ill patients. The increased solute transfer associated with the use of CRRT might enhance removal of amino acids, vitamins, catecholamines, and other solutes. As alluded to before, therapy downtime (the period when a prescribed CRRT has not run due to unplanned interruptions) should be carefully controlled, possibly limited and eventually compensated, because it might significantly impact dialysis delivery46–47. Following this path, it might be speculated that the quality of care and the specific dialysis monitoring is likely to be superior when a dialysis nurse is attending the treatment session48. In order to meet the safety requirement, the new generation of CRRT machines has been implemented with a strict safety profile limiting dangerous side effects of dialytic treatments. In any case, ICU staff training is mandatory before starting the routine utilization of such monitors.\n\nA synopsis of RRT prescription is also presented in Table 3.\n\nAbbreviations: CVC, central venous catheter; S-G, Swan Ganz catheter; EKG, electrocardiogram; CRRT, continuous renal replacement therapy; CVVHDF, continuous veno-venous hemodiafiltration; IHD, intermittent hemodialysis; MgPO4, magnesium phosphate.\n\nIn the specific setting of “weaning from RRT”, no good evidence exists at present and it is unlikely to be the case in the near future. Nevertheless, some insights may be gleaned from recent available literature. An interesting report from the Beginning and Ending Supportive Therapy for the Kidney (BEST Kidney) investigators described current practice for the discontinuation of CRRT in order to identify variables associated with successful discontinuation and whether the approach to discontinue CRRT therapy affected patient outcomes49. Statistical analysis identified urine output and creatinine as significant predictors of successful cessation. The predictive value of urine output was negatively affected by the use of diuretics. Risk factors for re-dialysis were also analyzed50: the 94 postoperative patients analyzed by these authors were considered free from RRT if after at least 30 days they did not require dialysis. Successful weaning from RRT was correlated with Sequential Organ Failure Assessment (SOFA) score, age, dialysis duration and, again, urine output. Interestingly, out of the patients who remained “RRT-free” for 5 days after RRT discontinuation, more than two-thirds (20) remained RRT-free for up to 30 days.\n\nAs a general recommendation, before weaning from RRT, physicians should wait for adequate urine output (without diuretic therapy) and optimized creatinine values. Once renal function appears close to the baseline or “pre-AKI” level, it seems reasonable to interrupt the treatment without any specific weaning protocol. Future trials, including the identification of new biomarkers, are needed to design novel weaning protocols.\n\n\nConclusions\n\nIn recent years, great technological improvements have been made in the manufacturing of extracorporeal circuits, rendering them easier to use, safer and more efficient for long-term support. Modern RRT systems can be managed in the ICU by one bedside nurse who is trained and experienced in circuit management, and it is now possible to treat patients for several weeks, or even months, without major complications. Thanks to technology development, the possibility of removing “waste products” is currently open to several molecules, including middle-sized ones, different from creatinine. It is now becoming a reality to integrate multiple devices into a single user-friendly machine for CO2 clearance, hemoperfusion, plasma-filtration and adsorption responding to different medical needs51–58. Finally, advances in information technology should allow the fully integrated extracorporeal blood purification system to be connected to all electronic therapeutic devices, from simple syringe pumps to CRRT machines, in order to ultimately lead to an ‘‘artificial organ’’ in a more complete sense59. In such a detailed and diversified technological world it is of utmost importance that communication among practitioners (physicians, nurses, technicians, researchers) is homogeneous and widely accepted. In light of this, nomenclature is a crucial aspect concerning RRT (please see Table 4 for a list of terminology and its significance). It is extremely important to avoid a sort of “Tower of Babel” effect by sharing a common language60.\n\nAbbreviations: Qb, blood flow; Qd, dialysis flow; Qf, ultrafiltration rate; UF, ultrafiltration; kD, kiloDaltons; HCO, high cut-off; IL, interleukin; AKI, acute kidney injury; ICU, intensive care unit.", "appendix": "Competing interests\n\n\n\nThe authors declare no personal or financial competing interests.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nMartin GS: Sepsis, severe sepsis and septic shock: changes in incidence, pathogens and outcomes. Expert Rev Anti Infect Ther. 2012; 10(6): 701–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRicci Z, Romagnoli S, Ronco C: Renal support. Minerva Anestesiol. 2011; 77(12): 1204–15. PubMed Abstract\n\nKramer P, Wigger W, Rieger J, et al.: [Arteriovenous hemofiltration: a new and simple method for treatment of over-hydrated patients resistant to diuretics]. Klin Wochenschr. 1977; 55(22): 1121–2. PubMed Abstract \| Publisher Full Text\n\nRonco C, Polaschegg HD: History and development of continuous renal replacement therapy. In: Critical Care Nephrology: Expert Consult – Edited by: Claudio Ronco MD, Rinaldo Bellomo MBBS(Hons) MD FRACP FCCP, John Kellum MD (Author). Philadelphia: Saunders Elseveier, 2009; 1323–1325.\n\nRicci Z, Bonello M, Salvatori G, et al.: Continuous renal replacement technology: from adaptive technology and early dedicated machines towards flexible multipurpose machine platforms. Blood Purif. 2004; 22(3): 269–276. PubMed Abstract \| Publisher Full Text\n\nRicci Z, Bellomo R, Ronco C: Renal Replacement Techniques: Descriptions, Mechanisms, Choices and Controverises. In: Critical Care Nephrology: Expert Consult – Edited by: Claudio Ronco MD, Rinaldo Bellomo MBBS(Hons) MD FRACP FCCP, John Kellum MD (Author). Philadelphia: Saunders Elseveier, 2009; 1136–41. Reference Source\n\nCole L, Bellomo R, Davenport P, et al.: Cytokine removal during continuous renal replacement therapy: an ex vivo comparison of convection and diffusion. Int J of Artif Organs. 2004; 27(5): 388–397. PubMed Abstract\n\nRicci Z, Ronco C, Bachetoni A, et al.: Solute removal during continuous renal replacement therapy in critically ill patients: convection versus diffusion. Crit Care. 2006; 10(2): R67. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYumoto M, Nishida O, Moriyama K, et al.: In vitro evaluation of high mobility group box 1 protein removal with various membranes for continuous hemofiltration. Ther Apher Dial. 2011; 15(4): 385–93. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nFriedrich JO, Wald R, Bagshaw SM, et al.: Hemofiltration compared to hemodialysis for acute kidney injury: systematic review and meta-analysis. Crit Care. 2012; 16(4): R146. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nWald R, Friedrich JO, Bagshaw SM, et al.: Optimal Mode of clearance in critically ill patients with Acute Kidney Injury (OMAKI)--a pilot randomized controlled trial of hemofiltration versus hemodialysis: a Canadian Critical Care Trials Group project. Crit Care. 2012; 16(5): R205. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nRicci Z, Bellomo R, Ronco C: Dose of dialysis in acute renal failure. Clin J Am Soc Nephrol. 2006; 1(3): 380–8. PubMed Abstract \| Publisher Full Text\n\nDellinger RP, Levy MM, Carlet JM, et al.: Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock: 2008. Crit Care Med. 2008; 36(1): 296–327. PubMed Abstract \| Faculty Opinions Recommendation\n\nVinsonneau C, Camus C, Combes A, et al.: Continuous venovenous haemodiafiltration versus intermittent haemodialysis for acute renal failure in patients with multiple-organ dysfunction syndrome: a multicentre randomised trial. Lancet. 2006; 368(9533): 379–85. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nKellum J, Palevsky PM: Renal support in acute kidney injury. Lancet. 2006; 368(9533): 344–5. PubMed Abstract \| Publisher Full Text\n\nGuérin C, Girard R, Selli JM, et al.: Intermittent versus continuous renal replacement therapy for acute renal failure in intensive care units: results from a multicenter prospective epidemiological survey. Intensive Care Med. 2002; 28(10): 1411–8. PubMed Abstract \| Publisher Full Text\n\nUehlinger DE, Jakob SM, Ferrari P, et al.: Comparison of continuous and intermittent renal replacement therapy for acute renal failure. Nephrol Dial Transplant. 2005; 20(8): 1630–7. PubMed Abstract \| Publisher Full Text\n\nSchortgen F, Soubrier N, Delclaux C, et al.: Hemodynamic tolerance of intermittent hemodialysis in critically ill patients: usefulness of practice guidelines. Am J Respir Crit Care Med. 2000; 162(1): 197–202. PubMed Abstract \| Publisher Full Text\n\nMarshall MR, Golper TA, Shaver MJ, et al.: Urea kinetics during sustained low-efficiency dialysis in critically ill patients requiring renal replacement therapy. Am J Kidney Dis. 2002; 39(3): 556–570. PubMed Abstract \| Publisher Full Text\n\nNaka T, Baldwin I, Bellomo R, et al.: Prolonged daily intermittent renal replacement therapy in ICU patients by ICU nurses and ICU physicians. Int J of Artif Organs. 2004; 27(5): 380–387. PubMed Abstract\n\nKumar VA, Craig M, Depner TA, et al.: Extended daily dialysis: A new approach to renal replacement for acute renal failure in the intensive care unit. Am J Kidney Dis. 2000; 36(2): 294–300. PubMed Abstract \| Publisher Full Text\n\nKielstein JT, Kretschmer U, Ernst T, et al.: Efficacy and cardiovascular tolerability of extended dialysis in critically ill patients: a randomized controlled study. Am J Kidney Dis. 2004; 43(2): 342–349. PubMed Abstract \| Publisher Full Text\n\nBaldwin I, Naka T, Koch B, et al.: A pilot randomised controlled comparison of continuous veno-venous haemofiltration and extended daily dialysis with filtration: effect on small solutes and acid–base balance. Intensive Care Med. 2007; 33(5): 830–5. PubMed Abstract \| Publisher Full Text\n\nRicci Z, Romagnoli S: Renal replacement therapy for critically ill patients: an intermittent continuity. Crit Care. 2014; 18(2): 115. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTan HK, Baldwin I, Bedllomo R: Continuous veno-venous hemofiltration without anticoagulation in high-risk patients. Intensive Care Med. 2000; 26(11): 1652–1657. PubMed Abstract \| Publisher Full Text\n\nFiaccadori E, Maggiore U, Rotelli C, et al.: Continuous haemofiltration in acute renal failure with prostacyclin as the sole anti-haemostatic agent. Intensive Care Med. 2002; 28(5): 586–593. PubMed Abstract \| Publisher Full Text\n\nKutsogiannis DJ, Gibney N, Stollery D, et al.: Regional citrate versus systemic heparin anticoagulation for continuous renal replacement in critically ill patients. Kidney Int. 2005; 67(6): 2361–2367. PubMed Abstract \| Publisher Full Text\n\nKim IB, Fealy N, Baldwin I, et al.: Insertion side, body position and circuit life during continuous renal replacement therapy with femoral vein access. Blood Purif. 2011; 31(1–3): 42–6. PubMed Abstract \| Publisher Full Text\n\nMonchi M, Berghmans D, Ledoux D, et al.: Citrate vs. heparin for anticoagulation in continuous venovenous hemofiltration: a prospective randomized study. Intensive Care Med. 2004; 30(2): 260–265. PubMed Abstract \| Publisher Full Text\n\nKDIGO AKI Work Group: KDIGO clinical practice guideline for acute kidney injury. Kidney Int (Suppl). 2012; 17: 1–138.\n\nJacobs R, Honoré PM, Bagshaw SM, et al.: Citrate Formulation Determines Filter Lifespan during Continuous Veno-Venous Hemofiltration: A Prospective Cohort Study. Blood Purif. 2015; 40(3): 194–202. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nPayen D, de Pont AC, Sakr Y, et al.: A positive fluid balance is associated with a worse outcome in patients with acute renal failure. Crit Care. 2008; 12(3): R74. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nRENAL Replacement Therapy Study Investigators, Bellomo R, Cass A, et al.: An observational study fluid balance and patient outcomes in the Randomized Evaluation of Normal vs. Augmented Level of Replacement Therapy trial. Crit Care Med. 2012; 40(6): 1753–60. PubMed Abstract \| Faculty Opinions Recommendation\n\nRonco C, Ricci Z: Renal replacement therapies: physiological review. Intensive Care Med. 2008; 34(12): 2139–46. PubMed Abstract \| Publisher Full Text\n\nGibney N, Cerda J, Davenport A, et al.: Volume management by renal replacement therapy in acute kidney injury. Int J Artif Organs. 2008; 31(2): 145–155. PubMed Abstract\n\nPonce D, Zorzenon Cde P, dos Santos NY, et al.: Early nephrology consultation can have an impact on outcome of acute kidney injury patients. Nephrol Dial Transplant. 2011; 26(10): 3202–6. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nRicci Z, Ronco C: Timing, dose and mode of dialysis in acute kidney injury. Curr Opin Crit Care. 2011; 17(6): 556–61. PubMed Abstract \| Publisher Full Text\n\nVaara ST, Reinikainen M, Wald R, et al.: Timing of RRT based on the presence of conventional indications. Clin J Am Soc Nephrol. 2014; 9(9): 1577–85. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nWald R, Adhikari NK, Smith OM, et al.: Comparison of standard and accelerated initiation of renal replacement therapy in acute kidney injury. Kidney Int. 2015; 88(4): 897–904. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nRonco C, Bellomo R, Homel P, et al.: Effects of different doses in continuous veno-venous haemofiltration on outcomes of acute renal failure: a prospective randomised trial. Lancet. 2000; 356(9223): 26–30. PubMed Abstract \| Publisher Full Text\n\nRENAL Replacement Therapy Study Investigators, Bellomo R, Cass A, et al.: Intensity of continuous renal-replacement therapy in critically ill patients. N Engl J Med. 2009; 361(17): 1627–38. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nVA/NIH Acute Renal Failure Trial Network, Palevsky PM, Zhang JH, et al.: Intensity of renal support in critically ill patients with acute kidney injury. N Engl J Med. 2008; 359(1): 7–20. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nUchino S, Bellomo R, Ronco C: Intermittent versus continuous renal replacement therapy in the ICU: impact on electrolyte and acid–base balance. Intensive Care Med. 2001; 27(6): 1037–43. PubMed Abstract \| Publisher Full Text\n\nPhu NH, Hien TT, Mai NT, et al.: Hemofiltration and peritoneal dialysis in infection-associated acute renal failure in Vietnam. N Engl J Med. 2002; 347(12): 895–902. PubMed Abstract \| Publisher Full Text\n\nElseviers MM, Lins RL, Van der Niepen P, et al.: Renal replacement therapy is an independent risk factor for mortality in critically ill patients with acute kidney injury. Crit Care. 2010; 14(6): R221. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nOudemans-van Straaten HM: Primum non nocere, safety of continuous renal replacement therapy. Curr Opin Crit Care. 2007; 13(6): 635–7. PubMed Abstract \| Publisher Full Text\n\nKleger GR, Fässler E: Can circuit lifetime be a quality indicator in continuous renal replacement therapy in the critically ill? Int J Artif Organs. 2010; 33(3): 139–46. PubMed Abstract \| Faculty Opinions Recommendation\n\nRicci Z, Benelli S, Barbarigo F, et al.: Nursing procedures during continuous renal replacement therapies: a national survey. Heart Lung Vessel. 2015; 7(3): 224–30. PubMed Abstract \| Free Full Text\n\nUchino S, Bellomo R, Morimatsu H, et al.: Discontinuation of continuous renal replacement therapy: a post hoc analysis of a prospective multicenter observational study. Crit Care Med. 2009; 37(9): 2576–2582. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nWu VC, Ko WJ, Chang HW, et al.: Risk factors of early redialysis after weaning from postoperative acute renal replacement therapy. Intensive Care Med. 2008; 34(1): 101–108. PubMed Abstract \| Publisher Full Text\n\nEarly Use of Polymyxin B Hemoperfusion in the Abdominal Sepsis 2 Collaborative Group: Polymyxin B hemoperfusion in clinical practice: the picture from an unbound collaborative registry. Blood Purif. 2014; 37(Suppl 1): 22–5. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nUchino S, Bellomo R, Goldsmith D, et al.: Super high flux hemofiltration: a new technique for cytokine removal. Intensive Care Med. 2002; 28(5): 651–655. PubMed Abstract \| Publisher Full Text\n\nLee PA, Weger GW, Pryor RW, et al.: Effects of filter pore size on efficacy of continuous arteriovenous hemofiltration therapy for Staphylococcus aureus-induced septicemia in immature swine. Crit Care Med. 1998; 26(4): 730–737. PubMed Abstract\n\nMorgera S, Haase M, Kuss T, et al.: Pilot study on the effects of high cutoff hemofiltration on the need for norepinephrine in septic patients with acute renal failure. Crit Care Med. 2006; 34(8): 2099–2104. PubMed Abstract \| Publisher Full Text\n\nPeng ZY, Kiss JE, Cortese-Hasset A, et al.: Plasma filtration on mediators of thrombotic microangiopathy: an in vitro study. Int J Artif Organs. 2007; 30(5): 401–406. PubMed Abstract\n\nFormica M, Inguaggiato P, Bainotti S, et al.: Coupled plasma filtration adsorption. Contrib Nephrol. 2007; 156: 405–410. PubMed Abstract\n\nBellomo R, Tetta C, Ronco C: Coupled plasma filtration adsorption. Intensive Care Med. 2003; 29(8): 1222–1228. PubMed Abstract \| Publisher Full Text\n\nRonco C, Brendolan A, Lonnemann G, et al.: A pilot study of coupled plasma filtration with adsorption in septic shock. Crit Care Med. 2002; 30(6): 1250–1255. PubMed Abstract \| Publisher Full Text\n\nRonco C, Ricci Z, De Backer D, et al.: Renal replacement therapy in acute kidney injury: controversy and consensus. Crit Care. 2015; 19: 146. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRonco C: The Charta of Vicenza. Blood Purif. 2015; 40(1): I–V. PubMed Abstract\n\nClark E, Molnar AO, Joannes-Boyau O, et al.: High-volume hemofiltration for septic acute kidney injury: a systematic review and meta-analysis. Crit Care. 2014; 18(1): R7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLivigni S, Bertolini G, Rossi C, et al.: Efficacy of coupled plasma filtration adsorption (CPFA) in patients with septic shock: a multicenter randomised controlled clinical trial. BMJ Open. 2014; 4(1): e003536. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "12081", "date": "25 Jan 2016", "name": "Neesh Pannu", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12082", "date": "25 Jan 2016", "name": "Patrick Honore", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12083", "date": "25 Jan 2016", "name": "Jay L. Koyner", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12084", "date": "25 Jan 2016", "name": "Ashita Tolwani", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-103
https://f1000research.com/articles/5-102/v1	25 Jan 16	{ "type": "Review", "title": "The underactive bladder: detection and diagnosis", "authors": [ "Nadir Osman", "Altaf Mangera", "Christopher Hillary", "Richard D. Inman", "Christopher R. Chapple", "Altaf Mangera", "Christopher Hillary", "Richard D. Inman", "Christopher R. Chapple" ], "abstract": "The inability to generate a voiding contraction sufficient to allow efficient bladder emptying within a reasonable time frame is a common problem seen in urological practice. Typically, the symptoms that arise are voiding symptoms, such as weak and slow urinary flow. These symptoms can cause considerable bother to patients and impact upon quality of life. The urodynamic finding of inadequate detrusor contraction has been termed detrusor underactivity (DUA). Although a definition is available for this entity, there are no widely accepted diagnostic criteria. Drawing parallels to detrusor overactivity and the overactive bladder, the symptoms arising from DUA have been referred to as the “underactive bladder” (UAB), while attempts to crystallize the definition of UAB are now ongoing. In this article, we review the contemporary literature pertaining to the epidemiology and etiopathogenesis of DUA as well as discuss the definitional aspects that are currently under consideration.", "keywords": [ "Underactive bladder", "detrusor underactivity", "chronic urinary retention" ], "content": "Introduction\n\nDetrusor underactivity (DUA) is a common bladder dysfunction observed in both men and women undergoing urodynamic studies for the assessment of lower urinary tract symptoms (LUTS). It is typically associated with voiding LUTS such as slow urinary flow and intermittency but may also result in storage and post-micturition LUTS. Despite being commonly encountered in routine clinical practice, DUA has been given relatively little attention in clinical and scientific research. Not only can DUA arise in bothersome symptoms but its occurrence may impact upon the outcome of treatments for other dysfunctions, such as bladder outlet obstruction and stress urinary incontinence. Recently, there has been renewed interest in DUA, the associated symptoms, and potential treatments. This article aims to review the recent literature regarding the epidemiology, etiopathogenesis, and diagnosis of DUA and its associated symptoms.\n\n\nDefinition\n\nA plethora of terms have been used to describe a bladder that does not contract efficiently. Terms such as detrusor areflexia, hypotonic bladder, atonic bladder, detrusor failure, impaired detrusor contractility, and chronic retention all reflect the lack of consensus and uncertainty as to the pathophysiological mechanisms at play. In an effort to standardize terminology, the International Continence Society report in 2002 used the term DUA to describe a urodynamic abnormality. This was defined as “a contraction of reduced strength and/or duration, resulting in prolonged bladder emptying and/or failure to achieve complete bladder emptying within a normal time span”1. This definition is necessarily vague on specifying the parameters for length and strength of contraction and prolonged voiding, as what is normal has not yet been established.\n\n\nSigns and symptoms\n\nThere is a lack of prospective studies correlating DUA and its associated clinical signs and symptoms. In clinical practice, the typical presentation is of voiding LUTS such as reduced flow, prolonged flow, hesitancy, and intermittency. After voiding, some patients may report a sensation of incomplete bladder emptying, which may be related to a raised post-voiding residual (PVR).\n\nOne could speculate that the presence of storage symptoms is related to the bladder sensory function in the individual patient, such that patients with poor or absent bladder sensation will have a reduced urge to pass urine, which will result in infrequent voiding. Anecdotally, some patients may report they lack any perception of their bladder emptying during the voiding process. Should these patients retain a large enough PVR, they may also experience incontinence, particularly during the night. This clinical picture is commonly termed chronic urinary retention (CUR). Patients with retained bladder sensation may void with a normal frequency pattern or have increased frequency, nocturia, and incontinence should the PVR be high enough.\n\nThere has been some debate about whether a symptom complex is associated with DUA, referred to by some as the underactive bladder (UAB), which is rather analogous to detrusor overactivity (DO) and the overactive bladder (OAB) symptom complex. This is certainly an attractive concept, as it could, in theory, allow patients to be diagnosed and treated on the basis of symptomatology. Nevertheless, there are clear difficulties in defining a specific set of symptoms that do not overlap significantly with LUTS due to benign prostatic enlargement (BPE). Clearly in women this is less likely to be problematic given the relatively rare occurrence of obstructive causes. A working definition for UAB was recently proposed by an expert consensus group: “…a symptom complex suggestive of detrusor underactivity and is usually characterised by prolonged urination time with or without a sensation of incomplete bladder emptying, usually with hesitancy, reduced sensation on filling, and a slow stream”2.\n\n\nEpidemiology\n\nGiven the inability to diagnose DUA without an invasive urodynamic study, it is unsurprising that there is little information on its prevalence on a population basis. There is no single non-invasive proxy measure (e.g. raised PVR, reduced urinary flow rate) that could reliably identify patients with the condition. Clinical series of patients with LUTS undergoing urodynamic studies offer the best available evidence as to the prevalence of DUA. In younger men (<50 years), prevalence is 9–28%, which rises to 48% in elderly men (>70 years)3. In elderly women, prevalence rates are between 12 and 45% and peak in the institutionalized elderly. Older frail patients often have co-existent DO, and this entity was originally described by Resnick and co-workers in 1987 as detrusor hyperactivity impaired contractility (DHIC)4.\n\n\nEtiopathogenesis\n\nGiven the diverse patient groups who can be affected by DUA, it is clear that the etiopathogenesis is likely to be multi-factorial. There seems to be an assumption in the literature that aging leads to a decline in detrusor contractile function. While plausible, it must be emphasized that there is no conclusive evidence that this is the case. The available data from animal and human studies is often quite contradictory.\n\nThe etiological factors resulting in DUA can be broadly classified as neurogenic or myogenic.\n\nAny pathological process that changes the normal structure and/or functioning of the extracellular matrix or the myocytes of the detrusor may cause a loss in the generation or transmission of a contraction. Factors that may affect the contractile activity of the myocytes include disruption of important cellular mechanisms (e.g. ion storage/exchange, excitation-contraction coupling, calcium storage, and energy generation). The implication of this is that even if efferent neuronal activity is normal, an impaired contraction will result5.\n\nSupporting the idea that myogenic factors may be important, morphological changes have been observed within both the normally aged and the diseased detrusor. Much of this work was conducted by Elbadawi, who suggested that specific ultrastructural features detected by electron microscopy were associated with aging and different bladder dysfunctions including DUA6–8. The specific pattern associated with DUA was described as the “degeneration” pattern, characterized by widespread disrupted myocytes with axonal degeneration6. What is not clear is whether detrusor myocyte disruption is in itself the cause of DUA or the result of an underlying pathological process.\n\nIntact bladder sensation is central to the voiding reflex. The afferent nerves monitor bladder volume during the storage phase and also during the voiding phase. Afferent nerves arising at the level of the urethra may also have a contributory role in perceiving flow through the urethra9,10. If afferent functioning is reduced, this may lead to a reduction in the strength or premature termination of the voiding reflex11.\n\nIt has long been held that DUA occurs as a secondary effect of prolonged bladder outlet obstruction (BOO). This assumption has been largely derived from work in animals, particularly the rodent, where partial BOO is induced by a constricting metal ring or ligature. The process by which DUA occurs has been widely described and includes three phases12,13:\n\n1. Following obstruction, there is increased bladder outlet resistance leading to bladder wall distension\n\n2. Compensatory hypertrophy and hyperplasia of the detrusor then occurs associated with a rise in tissue blood supply\n\n3. Initially, contractile function is maintained; however, after a variable period of time, contractility is dissipated with a reduction in bladder emptying ability (decompensation)\n\nRelating the insights from this work to humans is problematic, as the animals studied are usually young and female with an acute obstruction induced using a constricting ring. This is unlikely to be a good representation of the common clinical scenario of an elderly man with compressive benign prostatic obstruction. Interestingly, the largest longitudinal study available has shown that in man, prolonged BOO does not usually result in decompensation and DUA. In a group of 170 men with BOO demonstrated on pressure-flow studies, no significant deterioration in urodynamic parameters was observed at a mean follow up of 13.9 years (no change in pdet@Qmax [detrusor pressure at maximum flow] and a reduction in Qmax [maximum flow rate] of only 1 ml/s)14.\n\nDiabetic bladder dysfunction (DBD) (or diabetic cystopathy) is a well-recognized cause of DUA. Diabetes mellitus probably affects function through both a myogenic and a neurogenic mechanism leading to a decline in bladder emptying ability throughout the course of the disease15,16. It is traditionally thought that this predominately occurs through hyperglycemia-induced axonal degeneration and segmental demyelination leading to impaired afferent and efferent bladder function (autonomic neuropathy)17. Myogenic mechanisms are less clearly elucidated, though it is likely that changes in intercellular connections and excitability, intracellular signaling, and receptor density and distribution are implicated18.\n\n\nDiagnostic aspects\n\nCurrently, the only accepted modality for diagnosing DUA is an invasive pressure-flow test (or urodynamic study). However, there are no widely accepted criteria for diagnosing DUA. Most proposed methods relate to the measurement of the strength of the detrusor contraction.\n\nA detrusor contraction generates pressure and flow19; therefore, many authors have used two measurements to approximate contraction strength and thereby diagnose DUA: the maximal flow (Qmax) and the detrusor pressure at maximal flow (Pdet@Qmax). Most often a reduction in either is considered to be lower than the “normal” range. Historically, for men the normal ranges are derived from a series of men undergoing de-obstructive surgery20,21. In other groups, such as healthy men and women, in particular, the ranges are less well established22–24. Although relatively simple to apply, this method has two flaws. Firstly, it is likely to be an inaccurate method of determining the maximal pressure generated by the bladder due to the bladder outlet relation (BOR), the normal inverse correlation between detrusor pressure and urine flow during voiding25. Essentially, the BOR can be summed up as during voiding the pressure is highest when flow is lowest and vice versa, hence the pdet@Qmax represents the point of lowest bladder pressure during voiding. Secondly, the fact that the flow rate is also impacted upon by the degree of bladder outlet resistance is not considered. This is important because when detrusor pressure is low, the cause of low flow may also have its basis in obstruction (e.g. due to BPE) or alternatively a normal Qmax can result from reduced outlet resistance even if detrusor pressure is low (e.g. post-prostatectomy incontinence).\n\nIn order to gain a more accurate assessment of contraction strength, different methods of measuring isovolumetric bladder pressure have been introduced:\n\n1. Watts factor\n\nThis is an estimate of the power per unit area of the bladder surface that is generated by the detrusor. The formula for calculation is WF = [(pdet + a) (vdet + b) – ab]/2π where vdet is detrusor shortening velocity and a and b are fixed constants (a=25 cmH2O, b=6 mm/s) derived from experimental and clinical data26. The main pros of the WF are that it is not dependent on bladder volume26 and is not influenced by increased outlet resistance27. There are, however, no validated values for what is considered normal. Currently, the WF is little used in practice due to its complexity as well as the fact that it assesses only strength of contraction, rather than duration.\n\n2. Projected isovolumetric pressure (PIP)\n\nThe PIP was proposed by Schäfer, who used a straight line to represent the BOR and then “projected” back to the y-axis (pdet) from the point representing pdet@Qmax to obtain the isovolumetric pressure. This projection is calculated by the formula PIP = Pdet@Qmax + KQmax where K is a fixed constant representing the steepness of the angle of the BOR to x-a axis28. K is dependent on the specific population studied and differs between men and women29; in men it is usually taken as 5. The suggested groupings for PIP are as follows:\n\n>150 − strong contractility\n\n100 to 150 − normal contractility\n\n50 to 100 − weak contractility\n\n<50 − very weak contractility\n\nThere are two other reported variants of the PIP: the detrusor coefficient (DECO), which is the PIP divided by 100, and the bladder contractility index (BCI), which is essentially the same as PIP but with different groupings. The main advantage with these formulae is that they are easy and quick to use; however, they may overestimate PIP and have less test-retest reliability than measuring isovolumetric pressures directly30.\n\n3. Mechanical occlusion of flow\n\nThis entails the direct measurement of isovolumetric pressure by the mechanical obstruction of urine flow31. There are two methods for this measurement: (1) a stop test, the interruption of flow once it has already started, or (2) continuous occlusion test, where urine flow is impeded before and throughout the duration of the detrusor contraction. Stop tests can be voluntarily induced by the patient by contraction of the rhabdosphincter or alternatively the urodynamicist can impede flow through methods such as manual occlusion of the urethra. The voluntary stop test tends to underestimate pressure by about 20 cmH2O, which is likely to be attributable to a reflex detrusor inhibition brought on by sphincter contraction30,32. The voluntary test may not be possible in patients with sphincter weakness and in the elderly, whereas continuous occlusion can be painful and does not provide the opportunity for synchronous flow measurement.\n\n\nDetrusor contraction speed\n\nIf a bladder is slow to contract, then this could also potentially result in DUA. Cucchi and co-workers proposed the parameter of detrusor shortening velocity, as calculated by the formula vdet = Q/2[3/(V + Vt)/4π]0.66 where Q represents the flow rate (ml/s), V represents bladder volume (ml), and Vt represents the volume of non-contracting bladder wall tissue. They found that a reduction in shortening velocity was evident before a decline in Watt factor in both men33,34 and women35.\n\n\nAmbulatory urodynamics\n\nIt is not uncommon for a patient to be unable to void during a urodynamic study due to anxiety; this has been termed “bashful bladder”, which has been attributed to inadequate relaxation of the rhabdosphincter combined with reflex detrusor inhibition. This situation is usually identified by taking a good clinical history of previous voiding symptoms; however, if doubt persists, then an ambulatory urodynamic study may be helpful36,37.\n\n\nConclusions\n\nDUA remains a poorly characterized and poorly understood bladder dysfunction. Its clinical correlate, UAB, has recently been introduced into clinical usage, yet it remains unclearly defined. To progress the current situation, there is a need to investigate the common mechanisms by which patients develop DUA as well as to collect prospective data that can be used to correlate the symptoms and signs with an underlying urodynamic abnormality.", "appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nNadir Osman has received a grant from Astellas. Christopher R. Chapple has received grants and personal fees from Astellas, Allergan, and Recordati. All the other authors have no conflicts of interest to declare.\n\n\nReferences\n\nAbrams P, Cardozo L, Fall M, et al.: The standardisation of terminology of lower urinary tract function: report from the Standardisation Sub-committee of the International Continence Society. Neurourol Urodyn. 2002; 21(2): 167–78. PubMed Abstract \| Publisher Full Text\n\nChapple CR, Osman NI, Birder L, et al.: The underactive bladder: a new clinical concept? Eur Urol. 2015; 68(3): 351–3. PubMed Abstract \| Publisher Full Text\n\nOsman NI, Chapple CR, Abrams P, et al.: Detrusor underactivity and the underactive bladder: a new clinical entity? A review of current terminology, definitions, epidemiology, aetiology, and diagnosis. Eur Urol. 2014; 65(2): 389–98. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nResnick NM, Yalla SV, Laurino E: The pathophysiology of urinary incontinence among institutionalized elderly persons. N Engl J Med. 1989; 320(1): 1–7. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nBrierly RD, Hindley RG, McLarty E, et al.: A prospective controlled quantitative study of ultrastructural changes in the underactive detrusor. J Urol. 2003; 169(4): 1374–8. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nElbadawi A, Yalla SV, Resnick NM: Structural basis of geriatric voiding dysfunction. II. Aging detrusor: normal versus impaired contractility. J Urol. 1993; 150(5 Pt 2): 1657–67. PubMed Abstract\n\nElbadawi A, Yalla SV, Resnick NM: Structural basis of geriatric voiding dysfunction. III. Detrusor overactivity. J Urol. 1993; 150(5 Pt 2): 1668–80. PubMed Abstract\n\nElbadawi A, Yalla SV, Resnick NM: Structural basis of geriatric voiding dysfunction. IV. Bladder outlet obstruction. J Urol. 1993; 150(5 Pt 2): 1681–95. PubMed Abstract\n\nFeber JL, van Asselt E, van Mastrigt R: Neurophysiological modeling of voiding in rats: urethral nerve response to urethral pressure and flow. Am J Physiol. 1998; 274(5 Pt 2): R1473–81. PubMed Abstract\n\nBump RC: The urethrodetrusor facilitative reflex in women: results of urethral perfusion studies. Am J Obstet Gynecol. 2000; 182(4): 794–802; discussion 802–4. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nSuskind AM, Smith PP: A new look at detrusor underactivity: impaired contractility versus afferent dysfunction. Curr Urol Rep. 2009; 10(5): 347–51. PubMed Abstract \| Publisher Full Text\n\nLevin RM, Longhurst PA, Barasha B, et al.: Studies on experimental bladder outlet obstruction in the cat: long-term functional effects. J Urol. 1992; 148(3): 939–43. PubMed Abstract\n\nSaito M, Yokoi K, Ohmura M, et al.: Effects of partial outflow obstruction on bladder contractility and blood flow to the detrusor: comparison between mild and severe obstruction. Urol Int. 1997; 59(4): 226–30. PubMed Abstract \| Publisher Full Text\n\nThomas AW, Cannon A, Bartlett E, et al.: The natural history of lower urinary tract dysfunction in men: minimum 10-year urodynamic follow-up of untreated bladder outlet obstruction. BJU Int. 2005; 96(9): 1301–6. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nLifford KL, Curhan GC, Hu FB, et al.: Type 2 diabetes mellitus and risk of developing urinary incontinence. J Am Geriatr Soc. 2005; 53(11): 1851–7. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nLee WC, Wu HP, Tai TY, et al.: Effects of diabetes on female voiding behavior. J Urol. 2004; 172(3): 989–92. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nHill SR, Fayyad AM, Jones GR: Diabetes mellitus and female lower urinary tract symptoms: a review. Neurourol Urodyn. 2008; 27(5): 362–7. PubMed Abstract \| Publisher Full Text\n\nDaneshgari F, Liu G, Birder L, et al.: Diabetic bladder dysfunction: current translational knowledge. J Urol. 2009; 182(6 Suppl): S18–26. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGriffiths DJ: Editorial: bladder failure--a condition to reckon with. J Urol. 2003; 169(3): 1011–2. PubMed Abstract \| Publisher Full Text\n\nAbrams PH, Griffiths DJ: The assessment of prostatic obstruction from urodynamic measurements and from residual urine. Br J Urol. 1979; 51(2): 129–34. PubMed Abstract \| Publisher Full Text\n\nSchäfer W, Waterbär F, Langen PH, et al.: A simplified graphic procedure for detailed analysis of detrusor and outlet function during voiding. Neurourol Urodyn. 1989; 8: 405–7. Reference Source\n\nRosario DJ, Woo HH, Chapple CR: Definition of normality of pressure-flow parameters based on observations in asymptomatic men. Neurourol Urodyn. 2008; 27(5): 388–94. PubMed Abstract \| Publisher Full Text\n\nSchmidt F, Shin P, Jorgensen TM, et al.: Urodynamic patterns of normal male micturition: influence of water consumption on urine production and detrusor function. J Urol. 2002; 168(4 Pt 1): 1458–63. PubMed Abstract \| Publisher Full Text\n\nPfisterer MH, Griffiths DJ, Schaefer W, et al.: The effect of age on lower urinary tract function: a study in women. J Am Geriatr Soc. 2006; 54(3): 405–12. PubMed Abstract \| Publisher Full Text\n\nGriffiths DJ: The mechanics of the urethra and of micturition. Br J Urol. 1973; 45(5): 497–507. PubMed Abstract \| Publisher Full Text\n\nGriffiths DJ: Assessment of Detrusor Contraction Strength or Contractility. Neurourol Urodyn. 1991; 10(1): 1–18. Publisher Full Text\n\nLecamwasam HS, Yalla SV, Cravalho EG, et al.: The maximum watts factor as a measure of detrusor contractility independent of outlet resistance. Neurourol Urodyn. 1998; 17(6): 621–35. PubMed Abstract \| Publisher Full Text\n\nSchäfer W: Analysis of bladder-outlet function with the linearized passive urethral resistance relation, linPURR, and a disease-specific approach for grading obstruction: from complex to simple. World J Urol. 1995; 13(1): 47–58. PubMed Abstract \| Publisher Full Text\n\nGriffiths D: Detrusor contractility--order out of chaos. Scand J Urol Nephrol Suppl. 2004; 38(215): 93–100. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nTan TL, Bergmann MA, Griffiths D, et al.: Which stop test is best? Measuring detrusor contractility in older females. J Urol. 2003; 169(3): 1023–7. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nSullivan M, Yalla SV: Functional studies to assess bladder contractility. J urol Urogynäkol. 2007; 14(1): 7–10. Reference Source\n\nSullivan MP, DuBeau CE, Resnick NM, et al.: Continuous occlusion test to determine detrusor contractile performance. J Urol. 1995; 154(5): 1834–40. PubMed Abstract \| Publisher Full Text\n\nCucchi A, Quaglini S, Guarnaschelli C, et al.: Urodynamic findings suggesting two-stage development of idiopathic detrusor underactivity in adult men. Urology. 2007; 70(1): 75–9. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nCucchi A, Quaglini S, Rovereto B: Different evolution of voiding function in underactive bladders with and without detrusor overactivity. J Urol. 2010; 183(1): 229–33. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nCucchi A, Quaglini S, Rovereto B: Development of idiopathic detrusor underactivity in women: from isolated decrease in contraction velocity to obvious impairment of voiding function. Urology. 2008; 71(5): 844–8. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nRosario DJ, Chapple CR, Tophill PR, et al.: Urodynamic assessment of the bashful bladder. J Urol. 2000; 163(1): 215–20. PubMed Abstract \| Publisher Full Text\n\nvan Koeveringe GA, Rahnama'i MS, Berghmans BC: The additional value of ambulatory urodynamic measurements compared with conventional urodynamic measurements. BJU Int. 2010; 105(4): 508–13. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation" }	[ { "id": "12060", "date": "25 Jan 2016", "name": "Naoki Yoshimura", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12061", "date": "25 Jan 2016", "name": "Peter F.W.M Rosier", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe short commentary ‘The underactive bladder: detection and diagnosis’ discusses the topic from the perspective of symptoms, epidemiology, pathogenesis, and diagnostic aspects. The paragraphs on diagnostic aspects are a summary of urodynamic testing, more specifically: pressure-flow analysis. I assume that the words ‘…are typically associated with…’ in the introduction and the words ‘…typical presentation…’ in the signs and symptoms section, refer to the historical beliefs of professionals. The last paragraph of the signs and symptoms section mentions that there is a debate on whether there is a symptom complex associated with detrusor underactivity (the underactive bladder syndrome?). I agree with this statement and careful reading of the working definition cited from reference 2 in the commentary makes it obvious that the working definition of (the) bladder outflow obstruction (syndrome) would be exactly identical, if it was introduced nowadays. The elements of the working definition for UAB (as a signs and symptoms syndrome) are equal to those considered, both currently and traditionally, very relevant in men with benign prostatic enlargement1. I conclude that the underactive bladder syndrome as it is suggested here is not specific, like all LUTD-syndromes, and that, indeed, underactive detrusor (contraction during voiding) is a condition only assessable with a pressure-flow study.There is some observational clinical data on how the detrusor compensates for the growing prostate and the–by consequence–increase in bladder outflow obstruction, in addition to the animal studies referred to in the commentary, to explain the pathophysiology2. With the perspective of DU, this clinical study may allow two interpretations; first: the detrusor of aging men increases (maximum) power to compensate for the growing prostate and second: some detrusors are better able to compensate than others. The validity of the clinical observation2 was subsequently confirmed in a canine experiment3. If relevant neurogenic abnormalities are excluded on the basis of history and clinical examination, myogenic factors remain as the cause of detrusor underactivity, and based on biologic variance, some detrusors will be stronger than others and some will be better able to conquer challenges. The search for relevant amino-acids or other molecules will be important here if we want to discover ways to improve the symptomatically weak detrusors.Urodynamic diagnosis of detrusor contraction work or power during voiding is not yet standardized, and the commentary gives a neat overview of parameters that may prove to be useful. Especially objective parameters that will demonstrate predictive value and responsiveness to management will become relevant in the future.Cooperation with the patient is necessary to obtain a representative pressure-flow test, and the autonomic voiding reflex requires parasympathetic dominance in a–for the patient involved–stressful situation. This is even more important when the patient has a ‘bashful bladder’. I consider it valuable that this is introduced here in this context. Based on the relevance of this dysfunction, that will exist in a continuum from ‘low grade’ to ‘very troublesome‘, the new ICS Good Urodynamic Practices and Terms (that will be available in 2016) will introduce the terms (or diagnoses) ‘situational inability to void’ and ‘situational inability to void as usual’. Whether ambulatory urodynamics provides a reliable method to obtain a pressure-flow test remains speculative however, because of the lack of evidence.", "responses": [] } ]	1	https://f1000research.com/articles/5-102
https://f1000research.com/articles/5-98/v1	22 Jan 16	{ "type": "Research Note", "title": "Absence of diurnal variation in visceromotor response to colorectal distention in normal Long Evans rats", "authors": [ "Sara Botschuijver", "Zhumei Yu", "Olaf Welting", "Cathy Cailotto", "Andries Kalsbeek", "Rene van den Wijngaard", "Sara Botschuijver", "Zhumei Yu", "Olaf Welting", "Cathy Cailotto", "Andries Kalsbeek" ], "abstract": "Background: Enhanced colorectal sensitivity (i.e. visceral hypersensitivity) is thought to be a pathophysiological mechanism in irritable bowel syndrome (IBS). In healthy men a circadian variation in rectal perception to colonic distention was described. Disturbed day and night rhythms, which occur in shift work and trans meridian flights, are associated with the prevalence of IBS. This raises the question whether disruptions of circadian control are responsible for the observed pathology in IBS. Prior to investigating altered rhythmicity in relation to visceral hypersensitivity in a rat model for IBS, it is relevant to establish whether normal rats display circadian variation similar to healthy men. Methodology and findings: In rodents colorectal distension leads to reproducible contractions of abdominal musculature. We used quantification of this so called visceromotor response (VMR) by electromyography (EMG) to assess visceral sensitivity in rats. We assessed the VMR in normal male Long Evans rats at different time points of the light/dark cycle. Although a control experiment with male maternal separated rats confirmed that intentionally inflicted (i.e. stress induced) changes in VMR can be detected, normal male Long Evans rats showed no variation in VMR along the light/dark cycle in response to colorectal distension.Conclusions: In the absence of a daily rhythm of colorectal sensitivity in normal control rats it is not possible to investigate possible aberrancies in our rat model for IBS.", "keywords": [ "Circadian", "clock", "diurnal", "irritable bowel syndrome", "IBS", "perception", "rat", "visceral perception" ], "content": "Introduction\n\nIrritable bowel syndrome (IBS) is one of the most common functional gastrointestinal disorders and abdominal pain is the key contributing factor1. Up to 50% of patients have increased perception of gastrointestinal stimuli and this so called visceral hypersensitivity (assessed by rectal balloon distension) is considered a major pathophysiological mechanism2. Circadian variation in perception of rectal distention was described in healthy male volunteers3. The observed rhythmicity may relate to the autonomic nervous system being under the regime of the circadian clock4. This raises the question whether visceral hypersensitivity in IBS can be explained by disrupted circadian control of the autonomic nervous system. A subtle increase in sympathetic and decrease in parasympathetic nervous activity was observed in patients5 and complaints are known to show a daily variation, with higher pain scores early in the morning6. In addition, a disturbed day/night rhythm, which occurs in shift work and after transmeridian flights, is associated with the prevalence of IBS7,8. We wanted to investigate whether disrupted circadian control of the autonomic nervous system can explain post-stress hypersensitivity to colorectal distension observed in a rat model of IBS (i.e. the well validated maternal separation model9,10). However, before investigating possible aberrancies it was essential to confirm, in our experimental setting, earlier reported circadian variation of perception in normal ‘non-IBS’ rats11. In rodents, colorectal balloon distension leads to reproducible contractions of abdominal musculature (the visceromotor response [VMR]). The electromyographical (EMG) quantification of this response is often used to assess visceral sensitivity and changes thereof in rodents10,12. We used VMR quantification by EMG to assess possible circadian variation of visceral perception in Long Evans rats.\n\n\nMethods\n\nAll research was conducted in accordance with the institutional guidelines and approved by the Animal Ethical Committee of the AMC/University of Amsterdam (reference protocol number 100998).\n\n25 Long Evans rats (Harlan, Horst, Netherlands) were bred and housed at the animal facility of the Academic Medical Center (Amsterdam, Netherlands). Rats were housed in a 12 h light/12 h dark cycle (lights on at 07:00) under a constant temperature of 20±2C° and were provided with food and water ad libitum.\n\nTo avoid restraint stress during measurements, we previously validated and used radio-telemetry for assessing the VMR in freely moving rats12. The same methodology was used in the current investigations. In short, at a minimum age of 4 months, a telemetric transmitter with its two connected EMG electrodes (Physiotel Implant TA10AE-F20; Data Sciences International, St Paul, MN, USA) was implanted in the right side of the abdominal cavity. The electrodes were sutured in parallel into the left external abdominal oblique muscle 10 mm apart and 10 mm to the midline incision. After a postoperative recovery period of at least 10 days, the animals were subjected to colonic distention protocols. For this purpose, a latex balloon (Ultracover size 8F; International Medical Products BV, Zutphen, Netherlands) catheter was inserted 1 cm into the colon and fixed to the base of the tail under a short isoflurane anesthesia. After a 20 min recovery period, animals were placed in a macrolon cage (exact size of the receiver) that was positioned on top of the receiver. The receiver was linked to a Biopac MP100 data acquisition system (Biopac Systems Inc., Santa Barbara, CA, USA) and a personal computer via a raw data analog converter (Data Sciences International). Data were acquired with AcqKnowledge software (version 3.2.6, Biopac Systems Inc.). Colonic distention was achieved by slow manual inflation (5 s) of graded volumes of water (1.0, 1.5 and 2.0 mL) into the balloon using a syringe. Length and diameter of the balloon during a 2 mL maximum volume distention were 18 and 15 mm, respectively. All distensions lasted for 20 s and were separated by an 80 s rest in order to allow the EMG signal to return to baseline.\n\nOur earlier investigations indicated that repetitive distension sessions in normal male Long Evans rats (carried out at 09:00 on different days during a one month time period) result in equal VMR data13. Thus, inter distension-session interference is ruled out when repetitive measurements are carried out on different days. In the present investigations the VMR to colonic distension of 16 male nonhandled rats was measured at four different time points; 04:00, 08:00, 16:00, and 20:00. Experimental bias was avoided by stratifying rats into 4 equal groups: one group of four rats started sessions at 04:00, followed by 08:00, 16:00 and 20:00, another group started at 08:00 followed by 16:00, 20:00 and 04:00 etc. There was a minimum of 24 hours between distension sessions and all sessions within one group were completed within 15 days. To ascertain proper timing, not more than four animals per session were measured. During dark regime measurements, experiments were carried out under dim red light conditions and the experimental room was devoid of the regular tube light of the housing facility.\n\nIn adult male maternal separated Long Evans rats, acute stress is known to induce enhanced sensitivity to colorectal distension12–15. To assure that, when present, our methodology can accurately assess changes in VMR to colorectal distension, comparison of pre- and post-stress measurements in maternal separated rats was used as a positive control. Maternal separation was accomplished by placing the dams into another cage in another room for 180 minutes per day from postnatal day 2 to 14. During separation, cages were placed on a heating pad (30–34ºC) to help pups regulate normal body temperature. Pups were weaned on day 22 and subsequently raised in pairs of two until the age of 4 months when they were subjected to the experimental protocol. Earlier, we showed that post-stress hypersensitivity to distension will last for at least one month13. In line with the multiple distension sessions performed in the diurnal experiment (all four sessions carried out within a maximum timeframe of 15 days), colorectal distensions and concurrent EMG measurements were carried out pre- and 15 days post-stress. Rats were subjected to one single stress session by placing individual rats on top of a pedestal (8 · 8 · 10 cm) attached to the bottom of a plexiglass tank (25 · 25 · 45 cm). The tank was filled with fresh tap water at room temperature (21ºC) within 1 cm of the top of the pedestal and rats remained in the tank for 1 hour.\n\nData analysis was carried out similar to our earlier publications12,14,15. Each 20 s distension period and its preceding 20 s of baseline recording were extracted from the original raw EMG data file. After correction for movement and breathing, data were rectified and integrated. Absolute data sets were then obtained by subtracting the 20 s baseline recording from the 20 s distension result. Normalized data sets were then calculated from the absolute data by setting, in case of diurnal measurements, the 2 mL value of the 04:00 distension session, and in case of maternal separated rats the 2 mL value of the pre-stress distension session, at 100%. Area under the curve (AUC) of relative responses was calculated for individual rats and used to show possible changes in visceromotor response within groups. Relative response data were also used to evaluate possible changes on a per volume basis.\n\nStatistical analyses were performed using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, CA, USA). Diurnal variation in VMR (AUC as well as per volume results of the relative response) were evaluated by Friedman’s test. Pre- vs post-water avoidance data (AUC and per volume results of the relative response) of maternal separated rats were analysed by using Wilcoxon signed ranks test. All results are displayed as mean ± SEM and P<0.05 was considered significant.\n\n\nResults\n\nMean area under the curve ± SEM of the relative responses at 04:00, 08:00, 16:00 and 20:00 hrs in n=16 normal male Long Evans rats (Friedman’s test P=0.69) (A). Using the same data set but now depicting per volume relative responses on different time points (Friedman’s test P=0.55, 0.95 and 0.93; B, C and D respectively).\n\nPre- vs post-water avoidance area under the curve ± SEM of the relative response to distension (A). Same data set now depicted for per volume responses; increased post-stress response to distension at all 3 distension volumes (B). n=9, Wilcoxon’s test **P<0.01.\n\nWe assessed the VMR to distension in normal male Long Evans rats along four different time points of the light/dark cycle. Due to battery failure of its telemetric transmitter one animal could not be evaluated. Comparing area under the curve (AUC) of the relative response at the four time points we observed no daily variation (Figure 1A, n=15, Friedman’s test P=0.69). Since the AUC reflects the mean visceral sensitivity over a range of distension volumes, more subtle daily variations are perhaps better reflected when comparing results on a per volume basis. However, no significant differences were observed when comparing results for 1.0, 1.5 and 2.0 mL on the different time points (Figure 1B–D, Friedman’s test P=0.55, 0.95 and 0.93 respectively).\n\nWe next performed a control experiment to ascertain that, when present, our methodology was capable of detecting changes in VMR to distension. Similar to our earlier publications, male Long Evans pups were subjected to the maternal separation protocol and exposed to a one hour water avoidance stress at adult age. For reasons detailed in the methods section, distension sessions in the ‘diurnal variation assessment’ were carried out within a 15 day timeframe. Therefore, pre-water avoidance measurements in the maternal separation experiment were compared to measurements obtained 15 days post-water avoidance. Rats showed a significant post-stress increase in VMR to distension (Figure 2A, mean AUC ± SEM; 71.2 ± 2.4 vs 104 ± 4.8, Wilcoxon’s test P=0.004, pre- vs post-stress respectively). Figure 2B depicts the same data but now evaluated on a per volume basis; pre- vs post-water avoidance comparisons indicated significantly increased response to distension at all 3 distension volumes (P<0.01).\n\n\nDiscussion\n\nUnlike earlier observations in male Lewis rats11, our results indicate that male Long Evans rats do not experience daily variation of sensitivity to distension. Although EMG recordings of abdominal contractions were used as read-out in both rat studies, some differences in colonic distension methodology (volume vs isobaric distensions) are apparent and may explain the observed discrepancy. For our EMG recordings we used a radio-telemetry technique that we evaluated earlier, it allows for measurements in freely moving and non-fasted rats12. Performing measurements in freely moving rats lowers the amount of stress that is usually encountered when rats are restrained16 as is often the case during isobaric distensions. This is relevant because stress is a trigger for visceral hypersensitivity in patients as well as rats17,18. The downside of our approach is that freely moving rats easily expel from the colon the balloons suitable for isobaric distensions12. Thus, we performed less favored volume distensions which enable the use of balloons that will stay fit in the colon during rat movement, but cannot account for possible variations of colonic tone. The latter is an important difference with the methodology used by Gschossmann et al. who used barostat technology to perform isobaric distensions11. However, these results were obtained in Lewis rats that had to be subjected to an 18–24h fasting period prior to- as well as restraint during distensions; both fasting and restraint are known triggers for stress16,19. Moreover, compared to other rat strains Lewis rats are more stress sensitive and exhibit aberrant visceral pain sensitivity20. Especially because time of day of stress exposure is known to affect response levels21, it can be speculated that earlier observations in Lewis rats reflected methodology-induced rhythmicity of stress responses and accompanying changes in visceral sensitivity rather than diurnal variation in visceral sensitivity as such. Despite precautions taken, our own experiments also may have suffered from unforeseen/unnoticed methodology-induced bias affecting clock regulated mechanisms including diurnal sensitivity changes. Such bias could have been ruled out by establishing normal circadian release of an example hormone or the use of other readouts under the control of the biological clock. This however, would not have changed the end conclusion of these investigations.\n\nSimilar to the rat study discussed before, our results are also not in line with those obtained by Enck et al.3. These authors showed a daily rhythm of rectal perception in healthy male volunteers. In rats we evaluated four time points whereas seven were evaluated in the human study. Because of this, variations observed in men may have gone unnoticed in our male Long Evans rats. Further, volunteers were asked to refrain from eating starting at noon and received their first (light) meal at 00:45. Together with possible sleep deprivation (sleeping was allowed between 01:00 and 05:30 only) and continued unnatural conditions for study subjects (rectal balloon catheter remained present during entire timeframe of the study; 26 hours), the abnormal dietary pattern may have influenced study outcome in these healthy volunteers. Importantly, it is also possible that conflicting data arise because diurnal variation in visceral perception is present in healthy men but does not occur in normal Long Evans rats or cannot be properly detected in a robust rat model. Irrespective of these considerations, we have to conclude that in the absence of a circadian rhythm of colonic sensitivity in normal controls, our rat model and methodology are not suitable to investigate disturbed circadian rhythms in relation to visceral hypersensitivity in IBS.\n\n\nData availability\n\nF1000Research: Dataset 1. Excel datasheet showing raw EMG data acquired during colonic distensions, 10.5256/f1000research.7238.d11168922", "appendix": "Author contributions\n\n\n\nCC, AK and RvdW designed research; SB, ZY and OW conducted research; SB, ZY, OW and RvdW analyzed data; SB, ZY and RvdW wrote the paper. 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\nSB was supported by The Netherlands Organization for Health Research and Development (ZonMW), grant number 40-42600-98-071 (grant program ‘Meer Kennis met Minder Dieren’ aiming to publish solid negative data in animal research). ZY was sponsored by the China Exchange Programme of The Royal Netherlands Academy of Arts and Sciences (KNAW, project number 11CDDP005). OW was funded by a grant from the Dutch Organization for Scientific Research (NWO-VIDI).\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nDrossman DA, Morris CB, Schneck S, et al.: International survey of patients with IBS: symptom features and their severity, health status, treatments, and risk taking to achieve clinical benefit. J Clin Gastroenterol. 2009; 43(6): 541–550. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKeszthelyi D, Troost FJ, Masclee AA: Irritable bowel syndrome: methods, mechanisms, and pathophysiology. Methods to assess visceral hypersensitivity in irritable bowel syndrome. Am J Physiol Gastrointest Liver Physiol. 2012; 303(2): G141–54. PubMed Abstract \| Publisher Full Text\n\nEnck P, Kaiser C, Felber M, et al.: Circadian variation of rectal sensitivity and gastrointestinal peptides in healthy volunteers. Neurogastroenterol Motil. 2009; 21(1): 52–58. PubMed Abstract \| Publisher Full Text\n\nKalsbeek A, Yi CX, Cailotto C, et al.: Mammalian clock output mechanisms. Essays Biochem. 2011; 49(1): 137–51. PubMed Abstract \| Publisher Full Text\n\nManabe N, Tanaka T, Hata J, et al.: Pathophysiology underlying irritable bowel syndrome--from the viewpoint of dysfunction of autonomic nervous system activity. J Smooth Muscle Res. 2009; 45(1): 15–23. PubMed Abstract \| Publisher Full Text\n\nChang L, Lee OY, Naliboff B, et al.: Sensation of bloating and visible abdominal distension in patients with irritable bowel syndrome. Am J Gastroenterol. 2001; 96(12): 3341–7. PubMed Abstract \| Publisher Full Text\n\nHoogerwerf WA: Role of biological rhythms in gastrointestinal health and disease. Rev Endocr Metab Disord. 2009; 10(4): 293–300. PubMed Abstract \| Publisher Full Text\n\nNojkov B, Rubenstein JH, Chey WD, et al.: The impact of rotating shift work on the prevalence of irritable bowel syndrome in nurses. Am J Gastroenterol. 2010; 105(4): 842–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBarreau F, Ferrier L, Fioramonti J, et al.: New insights in the etiology and pathophysiology of irritable bowel syndrome: contribution of neonatal stress models. Pediatr Res. 2007; 62(3): 240–245. PubMed Abstract \| Publisher Full Text\n\nGreenwood-Van Meerveld B, Prusator DK, Johnson AC: Animal models of gastrointestinal and liver diseases. Animal models of visceral pain: pathophysiology, translational relevance, and challenges. Am J Physiol Gastrointest Liver Physiol. 2015; 308(11): G885–903. PubMed Abstract \| Publisher Full Text\n\nGschossmann JM, Buenger L, Adam B, et al.: Diurnal variation of abdominal motor responses to colorectal distension and plasma cortisol levels in rats. Neurogastroenterol Motil. 2001; 13(6): 585–9. PubMed Abstract \| Publisher Full Text\n\nWelting O, Van Den Wijngaard RM, De Jonge WJ, et al.: Assessment of visceral sensitivity using radio telemetry in a rat model of maternal separation. Neurogastroenterol Motil. 2005; 17(6): 838–845. PubMed Abstract \| Publisher Full Text\n\nVan Den Wijngaard RM, Stanisor OI, van Diest SA, et al.: Peripheral α-helical CRF (9-41) does not reverse stress-induced mast cell dependent visceral hypersensitivity in maternally separated rats. Neurogastroenterol Motil. 2012; 24(3): 274–82, e111. PubMed Abstract \| Publisher Full Text\n\nStanisor OI, van Diest SA, Yu Z, et al.: Stress-induced visceral hypersensitivity in maternally separated rats can be reversed by peripherally restricted histamine-1-receptor antagonists. PLoS One. 2013; 8(6): e66884. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVan Den Wijngaard RM, Stanisor OI, van Diest SA, et al.: Susceptibility to stress induced visceral hypersensitivity in maternally separated rats is transferred across generations. Neurogastroenterol Motil. 2013; 25(12): e780–e790. PubMed Abstract \| Publisher Full Text\n\nKramer K, Kinter L, Brockway BP, et al.: The use of radiotelemetry in small laboratory animals: recent advances. Contemp Top Lab Anim Sci. 2001; 40(1): 8–16. PubMed Abstract\n\nMayer EA, Naliboff BD, Chang L, et al.: V. Stress and irritable bowel syndrome. Am J Physiol Gastrointest Liver Physiol. 2001; 280(4): G519–G524. PubMed Abstract\n\nMoloney RD, O'Mahony SM, Dinan TG, et al.: Stress-induced visceral pain: toward animal models of irritable-bowel syndrome and associated comorbidities. Front Psychiatry. 2015; 6: 15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNowland MH, Hugunin KM, Rogers KL: Effects of short-term fasting in male Sprague-Dawley rats. Comp Med. 2011; 61(2): 138–44. PubMed Abstract \| Free Full Text\n\nO'Malley D, Julio-Pieper M, O'Mahony SM, et al.: Differential visceral pain sensitivity and colonic morphology in four common laboratory rat strains. Exp Physiol. 2014; 99(2): 359–67. PubMed Abstract \| Publisher Full Text\n\nKant GJ, Mougey EH, Meyerhoff JL: Diurnal variation in neuroendocrine response to stress in rats: plasma ACTH, beta-endorphin, beta-LPH, corticosterone, prolactin and pituitary cyclic AMP responses. Neuroendocrinology. 1986; 43(3): 383–90. PubMed Abstract \| Publisher Full Text\n\nBotschuijver S, Yu Z, Welting O, et al.: Dataset 1 in: Absence of diurnal variation in visceromotor response to colorectal distention in normal Long Evans rats. F1000Research. 2016. Data Source" }	[ { "id": "12045", "date": "29 Jan 2016", "name": "David Grundy", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 study using appropriate methodology providing a clear cut, if negative, data on diurnal variations in visceromotor response to colonic distension. The use of the maternally separated rat model provides validation of the model but raises the question of how diurnal variations may impact on stress induced hypersensitive VMR responses.", "responses": [] }, { "id": "12048", "date": "04 Feb 2016", "name": "Eamonn Quigley", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study is well done and the results are relatively straightforward. It would have been nice to have control responses on the VMR plots for reference so that one could gauge the magnitude of the response to colo-rectal distension, especially, as this was a novel distension protocol. From a methodological point of view their model is of interest because of its ability to allow the animals to move freely; comparisons with restrained animal results would be interesting.", "responses": [] }, { "id": "12101", "date": "15 Feb 2016", "name": "Siobhain O'Mahony", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nBotschuijver et al., present a well written study that is methodologically sound. They assess the diurnal variation (4 timepoints) in visceral sensitivity in Long Evans (male) rats. They also assess the impact of water avoidance stress on visceral sensitivity in maternally separated (male) rats. I thought it was still worthwhile assessing if there was a diurnal variation in the MS rats. The lack of difference in the control rats should not have deterred them from doing so as there are many studies where changes/differences are seen in man that can never be replicated in rat. Also the authors might want to consider a different strain of rat as different strains may be more responsive to the CRD procedure. Fig 1D-no error bar on 04:00 hours?", "responses": [] } ]	1	https://f1000research.com/articles/5-98
https://f1000research.com/articles/5-96/v1	22 Jan 16	{ "type": "Review", "title": "Recent advances in understanding urethral lichen sclerosus", "authors": [ "Altaf Mangera", "Nadir Osman", "Christopher R. Chapple", "Nadir Osman", "Christopher R. Chapple" ], "abstract": "Lichen sclerosus affecting the male genitalia is a poorly understood but potentially devastating condition. The natural history of the condition is beginning to be understood better with longer follow-up of patients. Recent long-term data suggest that circumcision for lichen sclerosus limited to the prepuce may not be curative as was once thought. In addition, surgical treatments should be followed up for longer periods as recurrences may occur after urethroplasty and perineal urethrostomy.", "keywords": [ "lichen sclerosus", "male lichen sclerosus", "inflammatory dermatosis" ], "content": "Introduction\n\nLichen sclerosus (LS) is a chronic inflammatory dermatosis with anogenital and extragenital presentations, the former of which is the most common. The epidermis and dermis are affected, and the aetiology remains unknown. In this review, we will consider LS affecting the male urethra and penile skin only. In this area, the skin typically becomes thickened and appears white, and the normal tissue architecture may be destroyed, earning it the outmoded name “balanitis xerotica obliterans”. LS may affect the prepuce alone or may include the external urethral meatus or distal urethra, and in severe cases the entire urethra may be involved. This review will update the reader on the latest evidence regarding aetiology and management of the various stages of LS.\n\n\nAetiology\n\nThe aetiology of LS remains unknown, but an autoimmune pathogenesis seems most likely. One study of vulval LS biopsies showed that oxidative stress at the cellular level may be responsible for the changes1. A larger study of female LS samples reported antibodies to matrix protein I in 75% of patients2. Another study, assessing 153 samples from men with and without LS, revealed a significant difference in serum extracellular matrix 1 antibody levels3. It is not clear what role the antibodies have in the genesis of LS. Another study compared male and female genital LS and reported higher numbers of CD4+ cells and a lower percentage of FOXP3+ lymphocytes in male LS, but both were higher than controls4. Interleukin-10-positive lymphocytes were lower in both compared with controls.\n\nA clinical review of 329 patients found other autoimmune disorders to exist less commonly in men with LS5. Men circumcised in childhood had the lowest risk, followed by men circumcised later. A viral aetiology has also been proposed as human papilloma virus was found in 50% of paediatric preputial tissue affected by LS by polymerase chain reaction6. Urinary pooling in the prepuce is also postulated to lead to LS7. It is also postulated that stricturing and obstruction of urine at the glans lead to the extravasation of urine into the glands of Littre, leading to the inflammation and spongiofibrosis seen in LS. The Koebner phenomenon, which occurs in damaged skin secondary to inflammation, also produces LS. So skin injury has been suggested as a possible trigger of LS in genetically predisposed people.\n\nLS in men may be limited to the prepuce or also include the glans or may be more aggressive, and spongiofibrosis may extend to affect the entire anterior urethra, which is much more common in adult men than children. Cystoscopically, the mucosa looks white or grey. The chronic inflammation has also been associated with squamous cell carcinoma, although the evidence regarding this is debatable8.\n\n\nManagement\n\nTo the best of our knowledge, there are no reported studies in the literature prospectively following the natural history of LS. Therefore, most information is gained from reports of LS management. In children, LS may be managed with steroid application. In a double-blind placebo-controlled trial, it was found that steroids could be used to reverse early-stage LS; however, even boys with early LS were completely resistant to steroid treatment9. Intralesional injection of triamcinolone has also been used to cause LS regression in mild cases but has a recurrence of 13%10. Circumcision is reported to be definitive in 96% of boys11. An interesting study of 99 patients having biopsies from different parts of the penis and urethra showed an interval of more than 10 years between circumcision and urethral involvement12. Also, progression of LS from meatus to the bulbar urethra was suggested to occur over the course of many years. Therefore, reporting a circumcision to be “curative” in 96% is possibly erroneous as most reports do not follow patients up for this long.\n\nIn a retrospective study of adult men, those who had steroid treatment of earlier disease were less likely to have recurrence compared to those with more extensive disease who required urethroplasty13. It is not clear from this study whether the men who required urethroplasty would have progressed to needing urethroplasty as their disease was more aggressive from the outset. It has also been reported that early and aggressive treatment of LS may help in preventing disease progression and recurrence, but again longer follow-up is required13.\n\nA recent study reported that 1 out of 5 boys who underwent circumcision subsequently required a meatotomy, and that almost all of these boys had previously not undergone a meatotomy14. The use of topical steroids was also associated with a reduced need for later meatotomy; however, it may have been that only boys with mild LS received steroids and therefore were less likely to recur at the outset. After meatotomy in LS, approximately 1 out of 4 patients will restenose and require further surgery15.\n\nAs it has been suggested that LS progresses from the meatus proximally toward the bulbar urethra over the course of many years12, it is not known whether the natural history of the condition can be changed by meatotomy or excision and grafting of a distal stricture. In LS a more extensive meatotomy (which leads to a hypospadiac meatus), including the use of grafts to try to reduce recurrence rates, has been advocated16. Malone has described a novel ventral and dorsal meatotomy with an inverted relaxing V incision with good results17.\n\nIn cases of more progressive disease affecting the urethra more proximally, urethroplasty is advocated18. Both one- and two-stage approaches for augmentation urethroplasty have been described, and the latter had a lower recurrence rate albeit with slightly shorter reported follow-up19. In either case, a skin graft should not be used, because of the high risk of recurrence, and instead an oral mucosa graft is advocated20. For single-stage repair, a urethral plate wider than 10 Fr is required and the disease should be mild. However, a recent study reported 90% success with the use of a one-stage urethroplasty with dorsal onlay oral mucosa grafts through a perineal incision21. The main complication of this approach was meatal stenosis. Morey has suggested that, in his experience, an extended meatotomy may be able to circumvent this problem16,22.\n\nIn severe cases, when the patient is unable or unwilling to have major urethral reconstruction, a perineal urethrostomy is a reasonable option23. Men will be required to sit to void but should retain sexual function. This is often considered to be the last line of management and has a risk of restenosis which requires further surgery24. Small case series have been reported of recurrence of LS in the edges of the skin surrounding the perineal urethrostomy and this was treated with potent steroid cream25. If this fails, further surgery is often required.\n\n\nConclusions\n\nLS is a little-understood condition of unknown aetiology. Observations have suggested an autoimmune pathology possibly related to chronic irritation of the urethra with urinary extravasation into the corpus spongiosum. If caught early, it has been shown to regress with steroids or potent anti-immune therapy. In the case of obstruction and irritation of the urethra, progression may occur. It is unclear whether early management of the obstruction arrests progression. Certainly patients presenting late may have involvement of the whole urethra.\n\nManagement involves a stepwise approach. The notion that circumcision is “curative” in more than 95% of patients with only foreskin involvement is probably inaccurate as recent data suggest a lag of more than 10 years for LS occurrence in the glans and urethra. Urethroplasty is feasible in these patients and should use oral mucosa. More data are required to determine whether there is a significant difference between the one- and two-stage approaches. Finally, a perineal urethrostomy may be appropriate for certain patients. With each of the management options, there is not one which can claim to “cure” the condition and therefore long-term follow-up is warranted.\n\n\nAbbreviation\n\nLS, lichen sclerosus.", "appendix": "Competing interests\n\n\n\nThe authors declared that they 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\nSander CS, Ali I, Dean D, et al.: Oxidative stress is implicated in the pathogenesis of lichen sclerosus. Br J Dermatol. 2004; 151(3): 627–35. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nOyama N, Chan I, Neill SM, et al.: Autoantibodies to extracellular matrix protein 1 in lichen sclerosus. Lancet. 2003; 362(9378): 118–23. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nEdmonds EV, Oyama N, Chan I, et al.: Extracellular matrix protein 1 autoantibodies in male genital lichen sclerosus. Br J Dermatol. 2011; 165(1): 218–9. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nGambichler T, Belz D, Terras S, et al.: Humoral and cell-mediated autoimmunity in lichen sclerosus. Br J Dermatol. 2013; 169(1): 183–4. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nEdmonds EV, Hunt S, Hawkins D, et al.: Clinical parameters in male genital lichen sclerosus: a case series of 329 patients. J Eur Acad Dermatol Venereol. 2012; 26(6): 730–7. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nDrut RM, Gómez MA, Drut R, et al.: Human papillomavirus is present in some cases of childhood penile lichen sclerosus: an in situ hybridization and SP-PCR study. Pediatr Dermatol. 1998; 15(2): 85–90. PubMed Abstract \| Publisher Full Text\n\nBunker CB, Shim TN: Male genital lichen sclerosus. Indian J Dermatol. 2015; 60(2): 111–7. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nPhilippou P, Shabbir M, Ralph DJ, et al.: Genital lichen sclerosus/balanitis xerotica obliterans in men with penile carcinoma: a critical analysis. BJU Int. 2013; 111(6): 970–6. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nKiss A, Csontai A, Pirót L, et al.: The response of balanitis xerotica obliterans to local steroid application compared with placebo in children. J Urol. 2001; 165(1): 219–20. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nWilkinson DJ, Lansdale N, Everitt LH, et al.: Foreskin preputioplasty and intralesional triamcinolone: a valid alternative to circumcision for balanitis xerotica obliterans. J Pediatr Surg. 2012; 47(4): 756–9. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nCelis S, Reed F, Murphy F, et al.: Balanitis xerotica obliterans in children and adolescents: a literature review and clinical series. J Pediatr Urol. 2014; 10(1): 34–9. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nBarbagli G, Mirri F, Gallucci M, et al.: Histological evidence of urethral involvement in male patients with genital lichen sclerosus: a preliminary report. J Urol. 2011; 185(6): 2171–6. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nTausch TJ, Peterson AC: Early aggressive treatment of lichen sclerosus may prevent disease progression. J Urol. 2012; 187(6): 2101–5. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nHomer L, Buchanan KJ, Nasr B, et al.: Meatal stenosis in boys following circumcision for lichen sclerosus (balanitis xerotica obliterans). J Urol. 2014; 192(6): 1784–8. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nSingh JP, Priyadarshi V, Goel HK, et al.: Penile lichen sclerosus: An urologist's nightmare! - A single center experience. Urol Ann. 2015; 7(3): 303–8. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nMorey AF, Lin HC, DeRosa CA, et al.: Fossa navicularis reconstruction: impact of stricture length on outcomes and assessment of extended meatotomy (first stage Johanson) maneuver. J Urol. 2007; 177(1): 184–7; discussion 187. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nMalone P: A new technique for meatal stenosis in patients with lichen sclerosus. J Urol. 2004; 172(3): 949–52. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nKulkarni S, Barbagli G, Kirpekar D, et al.: Lichen sclerosus of the male genitalia and urethra: surgical options and results in a multicenter international experience with 215 patients. Eur Urol. 2009; 55(4): 945–54. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nMangera A, Patterson JM, Chapple CR: A systematic review of graft augmentation urethroplasty techniques for the treatment of anterior urethral strictures. Eur Urol. 2011; 59(5): 797–814. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nVenn SN, Mundy AR: Urethroplasty for balanitis xerotica obliterans. Br J Urol. 1998; 81(5): 735–7. PubMed Abstract \| Publisher Full Text\n\nXu YM, Feng C, Sa YL, et al.: Outcome of 1-stage urethroplasty using oral mucosal grafts for the treatment of urethral strictures associated with genital lichen sclerosus. Urology. 2014; 83(1): 232–6. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nMorey AF: Re: outcome of 1-stage urethroplasty using oral mucosal grafts for the treatment of urethral strictures associated with genital lichen sclerosus. J Urol. 2014; 191(6): 1814. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nStewart L, McCammon K, Metro M, et al.: SIU/ICUD Consultation on Urethral Strictures: Anterior urethra-lichen sclerosus. Urology. 2014; 83(3 Suppl): S27–30. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nLopez JC, Gomez EG, Carrillo AA, et al.: Perineostomy: the last oportunity. Int Braz J Urol. 2015; 41(1): 91–8; discussion 99–100. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nShim TN, Andrich DE, Mundy AR, et al.: Lichen sclerosus associated with perineal urethrostomy. Br J Dermatol. 2014; 170(1): 222–3. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation" }	[ { "id": "12042", "date": "22 Jan 2016", "name": "Andrew C. Peterson", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12043", "date": "22 Jan 2016", "name": "Lee C. Zhao", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12044", "date": "22 Jan 2016", "name": "Tang Ngee Shim", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-96
https://f1000research.com/articles/5-95/v1	22 Jan 16	{ "type": "Review", "title": "Innovative Applications of Robotic Surgery: Renal Allograft and Autologous Transplantation", "authors": [ "Jason Lee", "Michael Ordon", "Michael Ordon" ], "abstract": "Robotic surgery has enabled surgeons to offer more patients a minimally invasive surgical option in the management of their complex diseases. While renal transplantation is associated with significant improvements in quantity and quality of life for most end-stage renal disease (ESRD) patients, it is also not devoid of its surgical risks and potential morbidities. Robotic-assisted kidney transplantation is a recently described, innovative application of the robotic surgery platform, and early experiences suggest that it is associated with comparable graft function and lower rates of complications.Urinary tract obstruction, though less common than ESRD, can be a serious threat to renal function. Severe ureteric stricture disease can represent a clinically complex problem requiring major reconstructive surgery. Completely intra-corporeal robotic renal auto-transplantation is another innovative application of the robotic surgery platform and represents a significant advancement in urologic surgery. Initial reports of this procedure demonstrate safety, feasibility, and excellent renal function outcomes.", "keywords": [ "Kidney Transplantation", "Renal Allograft Transplantation", "Robotic Renal Transplantation", "Renal Autologous Transplantation", "Robotic Surgery" ], "content": "\n\nRobotic-assisted laparoscopic surgery has gained widespread adoption globally within the field of urology. Technologic advancements have led to improved surgical dexterity and vision and, combined with a more facile learning curve than pure laparoscopic surgery, the robotic surgery platform has enabled more surgeons to perform minimally invasive surgical (MIS) procedures. Moreover, not only has robotic surgery afforded more patients access to a MIS option, it has also enabled urologists to perform extremely complex extirpative and reconstructive procedures, traditionally performed in an open fashion due to their technically challenging nature, with a minimally invasive approach.\n\nThe first successful kidney transplant was reported in 19561 and since that time renal transplantation has changed the lives of millions of patients with end-stage renal disease (ESRD) through improved quantity as well as quality of life2. With the success of living donor renal transplantation programs, outcomes for patients with ESRD have improved even further.\n\nUnlike healthy living donors, most kidney transplant recipients are, by the very nature of their disease, high-risk surgical candidates. While the long-term benefits of renal transplantation have been clearly documented3–5, there are tangible peri-operative risks for ESRD patients undergoing transplantation that need to be weighed against these long-term benefits.\n\nThe advantages of MIS have been well documented in the literature and include less post-operative pain, quicker recovery time, lower rates of incisional complications, less blood loss, and better cosmesis6–8. Level 1 evidence supports a minimally invasive approach for donor nephrectomy9 in a patient population that is healthy and highly screened and selected. But for the high-risk ESRD recipients, for whom the peri-operative advantages of MIS would be evermore beneficial, a more morbid open surgical approach to renal transplantation still remains the gold standard.\n\nAs robotic surgery has become more pervasive and mainstream, and with improved experience and training, it has allowed highly skilled and innovative surgeons to now offer MIS options for these higher-risk ESRD patients.\n\nThe first reported use of robotics to perform a kidney transplant was in 200210; however, this was in essence a hybrid operation whereby the da Vinci® (Intuitive Surgical Inc, USA) robotic surgical platform was used to perform a conventional open deceased donor kidney transplant through a large open incision.\n\nThe first true robotic-assisted laparoscopic kidney transplant (RAKT) was reported by the group from University of Illinois at Chicago in 201011. Using the da Vinci® robot, Giulianotti et al. performed a deceased donor RAKT utilizing a 7 cm peri-umbilical incision through which the graft was introduced into the peritoneum. Total operative time was 223 minutes, with warm ischemia time of 50 minutes. The following year, Boggi et al. published the first successful European RAKT, but described a slightly different technique12. Rather than a peri-umbilical incision, the authors utilized a 7 cm Pfannenstiel incision. While the vascular anastomoses were performed entirely robotically, the ureterovesical anastomosis was performed in an open fashion through the Pfannenstiel incision. Total operative time was 154 minutes, with 51 minutes of warm ischemia. In 2015, Doumerc et al. described another novel approach to RAKT, in which they utilized a vaginal incision to introduce the renal graft into the peritoneum, transvaginally, inside a sterile Endobag13. Similar to the other case reports, mean operative time was 200 minutes and anastomotic time was 55 minutes. While only feasible in female ESRD recipients, this novel transvaginal technique eliminates the need for a larger abdominal incision, further accentuating the minimally invasive nature of RAKT and thereby perhaps further decreasing the morbidity of renal transplantation surgery. Building on their novel technique, later that year, Doumerc and Sallusto reported the first pure robot-assisted approach to living donor kidney transplantation utilizing the transvaginal technique for both the donor and the recipient surgeries14.\n\nWhile these pioneering surgeons demonstrated the safety and feasibility of this innovative procedure, the reported RAKT techniques did not involve intra-corporeal cold perfusion of the graft. In addition, these early reports had longer warm ischemic times than commonly seen with open kidney transplantation. How this slight increase in warm ischemia truly impacts long-term graft function is yet unknown, but clearly delineates an opportunity for improvement.\n\nIn 2014, two separate publications from the same authorship group reported on a RAKT case series that utilized a new technique allowing for intracorporeal regional hypothermia of the graft15,16. The authors used a peri-umbilical incision as well, but described the use of a novel gauze-jacket filled with ice-slush. This served to minimize warm ischemia and also allowed for atraumatic handling of the graft. Additional ice-slush was introduced into the peritoneum to cover the graft once it had been placed into the peritoneum. Utilizing this technique, the mean operative time in the case series was 214 minutes, with a mean “warm” ischemia time of 47 minutes15. The authors cited that while there was clear evidence for the feasibility and safety of this innovative procedure, comparative studies were still required to determine the cost-effectiveness of RAKT over conventional open kidney transplantation.\n\nIn line with existing comparative literature evaluating laparoscopic and open surgery, initial experiences at various institutions around the world have now demonstrated lower complication rates for RAKT in comparison to similar open renal transplant cohorts15,17,18. With comparative ischemia times and the ability to cool the graft intracorporeally, graft function outcomes also seem to be at least equivalent to traditional open renal transplantation15,17.\n\nAmong a cohort of morbidly obese ESRD patients, Oberholzer et al. demonstrated that RAKT was associated with better outcomes in comparison to conventional open kidney transplantation. The authors pointed out that with improved outcomes after RAKT in the morbidly obese patient population, it may result in increased access to life-saving transplantation surgery for patients who may have otherwise been deemed unsuitable candidates due to their increased peri-operative risks17.\n\nThese initial RAKT cohorts still represent a small sample size, and as such comparative assessment with larger cohorts will be necessary in time to further support the initial findings, which are very encouraging.\n\nOne of the most common criticisms regarding the role of RAKT is the fact that an open incision, at least the size of the graft, is required regardless in order to introduce the allograft kidney into the peritoneal space, so why not simply use a slightly larger incision and do the surgery in a more conventional manner. RAKT supporters argue that any minimization of incision length, particularly in the immunosuppressed, ESRD patient population, can decrease the not-insignificant surgical incision-related complication rates seen in these patients. This is particularly true among obese transplant recipients, who are prone to much higher rates of incisional complications17.\n\nRenal autologous transplantation (ReATx) was first reported in 1963 and represents a viable option in the management of long or severe upper ureteric strictures19. While much less commonly performed than allograft renal transplantation, ReATx is a definitive surgical option that allows for the preservation of renal function, whether imperative or elective, while reconstituting normal urinary drainage. The morbidity of ReATx is not inconsequential, however, as it involves two very distinct and complex procedures.\n\nThe laparoscopic approach to the management of many different urologic diseases has now become commonplace. With this, the morbidity of ReATx has significantly improved as well and, currently, the most common approach to ReATx is laparoscopic donor nephrectomy followed by open ReATx20,21. This approach has demonstrated excellent outcomes and is considered by many to be the gold standard approach.\n\nUnlike deceased donor or living donor renal transplantation surgery, for patients deemed suitable for ReATx, the allograft kidney is already located intra-corporeally. As such, if one were to be able to perform both distinct stages of the ReATx (i.e. donor nephrectomy and auto-transplantation), utilizing a MIS technique while maintaining the allograft intra-corporeally, the morbidity associated with a large surgical incision would be avoided completely. The ability to do this adds extreme technical complexity and would necessitate not only intra-corporeal preparation of the graft but completely intra-corporeal perfusion, and hypothermia as well.\n\nTaking their experience with RAKT one step further, Abaza and colleagues reported the first ever completely intra-corporeal robotic-assisted ReATx surgery in 201422. This truly innovative application of the robotic surgery platform allowed for the management of severe ureteric stricture disease without the allograft ever having to be removed from the patient. Total operative time was 425 minutes and total ischemic time was 127 minutes. While this pioneering report demonstrated the safety and feasibility of robotic-assisted ReATx, the described technique was associated with longer ischemia than one would encounter with the conventional approach to ReATx: laparoscopic donor nephrectomy, ex vivo preparation, and open auto-transplantation9,23.\n\nBuilding on their seminal work, we reported the first completely intra-corporeal robotic-assisted ReATx in Canada24 but modified the technique utilized by Abaza and colleagues22 in an attempt to minimize renal ischemia. Similar to their described technique, intra-corporeal renal perfusion with cooled HTK and normal saline solution was initiated immediately after donor nephrectomy using a perfusion cannula inserted through a 12 mm assistant port. By altering the technique from a two-stage to a three-stage procedure, however, we were able to decrease the total operative time to 390 minutes and, more significantly, we were able to complete the surgery with only 79 minutes of ischemia (4 minutes of warm ischemia, 48 minutes of cold ischemia, and 27 minutes of re-warming time), which is more comparable to what is seen with conventional ReATx surgery.\n\nWhile these two initial reports have demonstrated the safety and feasibility of completely intra-corporeal robotic ReATx, both case reports involved kidneys with relatively straightforward renal vasculature. With any increased complexity (e.g. two or more renal arteries), vascular reconstruction without removal of the kidney ex vivo would necessitate significant alterations in technique. We are currently working on developing such techniques that would allow for the allograft to remain intra-corporeally, while simultaneously minimizing renal ischemia during vascular reconstruction and auto-transplantation.\n\n\nConclusions\n\nRobotic surgery has enabled surgeons to offer more patients a minimally invasive surgical option in the management of their complex diseases. Robotic-assisted kidney transplantation and completely intra-corporeal robotic renal auto-transplantation are recent innovative applications of the robotic surgery platform and represent significant advancements in urologic surgery. These novel applications of robotic surgery will hopefully result in improved patient outcomes while simultaneously achieving lower patient morbidity.", "appendix": "Competing interests\n\n\n\nThe authors declared that they 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\nGuild WR, Harrison JH, Merrill JP, et al.: Successful homotransplantation of the kidney in an identical twin. Trans Am Clin Climatol Assoc. 1955–1956; 67: 167–73. PubMed Abstract \| Free Full Text\n\nHiggins MR, Grace M, Dossetor JB: Survival of patients treated for end-stage renal disease by dialysis and transplantation. Can Med Assoc J. 1977; 117(8): 880–3. PubMed Abstract \| Free Full Text\n\nSuthanthiran M, Strom TB: Renal transplantation. N Engl J Med. 1994; 331(6): 365–76. PubMed Abstract \| Publisher Full Text\n\nVollmer WM, Wahl PW, Blagg CR: Survival with dialysis and transplantation in patients with end-stage renal disease. N Engl J Med. 1983; 308(26): 1553–8. PubMed Abstract \| Publisher Full Text\n\nWolfe RA, Ashby VB, Milford EL, et al.: Comparison of mortality in all patients on dialysis, patients on dialysis awaiting transplantation, and recipients of a first cadaveric transplant. N Engl J Med. 1999; 341(23): 1725–30. PubMed Abstract \| Publisher Full Text\n\nGill IS, Matin SF, Desai MM, et al.: Comparative analysis of laparoscopic versus open partial nephrectomy for renal tumors in 200 patients. J Urol. 2003; 170(1): 64–8. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nParker PA, Swartz R, Fellman B, et al.: Comprehensive assessment of quality of life and psychosocial adjustment in patients with renal tumors undergoing open, laparoscopic and nephron sparing surgery. J Urol. 2012; 187(3): 822–6. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nNicholson ML, Kaushik M, Lewis GR, et al.: Randomized clinical trial of laparoscopic versus open donor nephrectomy. Br J Surg. 2010; 97(1): 21–8. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nDols LF, Kok NF, Ijzermans JN: Live donor nephrectomy: a review of evidence for surgical techniques. Transpl Int. 2010; 23(2): 121–30. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nHoznek A, Zaki SK, Samadi DB, et al.: Robotic assisted kidney transplantation: an initial experience. J Urol. 2002; 167(4): 1604–6. PubMed Abstract\n\nGiulianotti P, Gorodner V, Sbrana F, et al.: Robotic transabdominal kidney transplantation in a morbidly obese patient. Am J Transplant. 2010; 10(6): 1478–82. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nBoggi U, Vistoli F, Signori S, et al.: Robotic renal transplantation: first European case. Transpl Int. 2011; 24(2): 213–8. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nDoumerc N, Roumiguié M, Rischmann P, et al.: Totally Robotic Approach with Transvaginal Insertion for Kidney Transplantation. Eur Urol. 2015; 68(6): 1103–4. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nDoumerc N, Beauval JB, Rostaing L, et al.: A new surgical area opened in renal transplantation: a pure robot-assisted approach for both living donor nephrectomy and kidney transplantation using transvaginal route. Transpl Int. 2016; 29(1): 122–3. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nMenon M, Sood A, Bhandari M, et al.: Robotic kidney transplantation with regional hypothermia: a step-by-step description of the Vattikuti Urology Institute-Medanta technique (IDEAL phase 2a). Eur Urol. 2014; 65(5): 991–1000. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nAbaza R, Ghani KR, Sood A, et al.: Robotic kidney transplantation with intraoperative regional hypothermia. BJU Int. 2014; 113(4): 679–81. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nOberholzer J, Giulianotti P, Danielson KK, et al.: Minimally invasive robotic kidney transplantation for obese patients previously denied access to transplantation. Am J Transplant. 2013; 13(3): 721–8. PubMed Abstract \| Publisher Full Text \| Free Full Text \| F1000 Recommendation\n\nTsai MK, Lee CY, Yang CY, et al.: Robot-assisted renal transplantation in the retroperitoneum. Transpl Int. 2014; 27(5): 452–7. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nHardy JD: High ureteral injuries. Management by autotransplantation of the kidney. JAMA. 1963; 184(2): 97–101. PubMed Abstract \| Publisher Full Text\n\nAzhar B, Patel S, Chadha P, et al.: Indications for renal autotransplant: an overview. Exp Clin Transplant. 2015; 13(2): 109–14. PubMed Abstract \| Publisher Full Text\n\nTran G, Ramaswamy K, Chi T, et al.: Laparoscopic Nephrectomy with Autotransplantation: Safety, Efficacy and Long-Term Durability. J Urol. 2015; 194(3): 738–43. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nGordon ZN, Angell J, Abaza R: Completely intracorporeal robotic renal autotransplantation. J Urol. 2014; 192(5): 1516–22. PubMed Abstract \| Publisher Full Text \| F1000 Recommendation\n\nTreat EG, Miller ET, Kwan L, et al.: Outcomes of shipped live donor kidney transplants compared with traditional living donor kidney transplants. Transpl Int. 2014; 27(11): 1175–82. PubMed Abstract \| Publisher Full Text\n\nLee JY, Alzahrani T, Ordon M: Intra-corporeal robotic renal auto-transplantation. Can Urol Assoc J. 2015; 9(9–10): E748–9. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "11938", "date": "22 Jan 2016", "name": "Rajesh Ahlawat", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "11939", "date": "22 Jan 2016", "name": "Federico Sallusto", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] } ]	1	https://f1000research.com/articles/5-95
https://f1000research.com/articles/4-1279/v1	17 Nov 15	{ "type": "Research Article", "title": "A three-dimensional model of the human blood-brain barrier to analyse the transport of nanoparticles and astrocyte/endothelial interactions", "authors": [ "Peddagangannagari Sreekanthreddy", "Radka Gromnicova", "Heather Davies", "James Phillips", "Ignacio A. Romero", "David Male", "Peddagangannagari Sreekanthreddy", "Radka Gromnicova", "Heather Davies", "James Phillips", "Ignacio A. Romero" ], "abstract": "The aim of this study was to develop a three-dimensional (3D) model of the human blood-brain barrier in vitro, which mimics the cellular architecture of the CNS and could be used to analyse the delivery of nanoparticles to cells of the CNS. The model includes human astrocytes set in a collagen gel, which is overlaid by a monolayer of human brain endothelium (hCMEC/D3 cell line). The model was characterised by transmission electron microscopy (TEM), immunofluorescence microscopy and flow cytometry. A collagenase digestion method could recover the two cell types separately at 92-96% purity. Astrocytes grown in the gel matrix do not divide and they have reduced expression of aquaporin-4 and the endothelin receptor, type B compared to two-dimensional cultures, but maintain their expression of glial fibrillary acidic protein. The effects of conditioned media from these astrocytes on the barrier phenotype of the endothelium was compared with media from astrocytes grown conventionally on a two-dimensional (2D) substratum. Both induce the expression of tight junction proteins zonula occludens-1 and claudin-5 in hCMEC/D3 cells, but there was no difference between the induced expression levels by the two media. The model has been used to assess the transport of glucose-coated 4nm gold nanoparticles and for leukocyte migration. TEM was used to trace and quantitate the movement of the nanoparticles across the endothelium and into the astrocytes. This blood-brain barrier model is very suitable for assessing delivery of nanoparticles and larger biomolecules to cells of the CNS, following transport across the endothelium.", "keywords": [ "in vitro model", "blood-brain barrier", "three-dimensional", "endothelium", "astrocytes", "nanoparticles" ], "content": "Introduction\n\nThe human blood brain barrier presents a major challenge for the pharmaceutical industry, and a number of cell culture systems have been developed which model different aspects of the barrier (Naik & Cucullo, 2012; Ogunshola, 2011). In vivo, the barrier is formed by specialised brain endothelial cells that have continuous tight junctions, low pinocytotic activity and a set of enzymes and ABC-transporters that metabolise or exclude many xenobiotics (Sarkadi et al., 2006; Wolburg & Lippoldt, 2002). The function and structure of the brain endothelium is supported by astrocytes; astrocytic foot processes ensheath the abluminal side of the endothelium and form a double basal lamina, with the endothelium (Abbott et al., 2010). Several tissue culture models of the barrier have been developed and each one has some of the characteristics seen in vivo, although the barrier is generally not as tight as in vivo (Butt et al., 1990). Hence, each model is suitable for specific purposes, although none of the models is ideal for all applications (Naik & Cucullo, 2012). One aim of this study was to develop a model of the human blood-brain barrier, which could be used to study nanoparticle movement across brain endothelium and localisation in glial cells. This model might also be used to study interactions between brain endothelium and astrocytes.\n\nOne problem in the development of barrier models is the limited availability of primary human brain endothelium, although this has been partly compensated by the development of stem-cell derived brain endothelium (Lippmann et al., 2012). Primary endothelium at low passage has high resistance tight junctions, and expresses appropriate transporters, but the barrier phenotype tends to decline in extended culture (Lyck et al., 2009). The availability of the human brain endothelial line hCMEC/D3 (Weksler et al., 2005) has been used to develop several in vitro models (Weksler et al., 2013). These cells do not form a strong barrier for molecules <4kDa under static conditions, although the transendothelial electrical resistance (TEER) is enhanced in flow-based conditions (Cucullo et al., 2008).\n\nBarrier models which include human astrocytes have generally cultured the endothelium on the upper surface of filters in transwell inserts, and the astrocytes are either cultured on the lower surface of the filter or on the base of the culture well (Naik & Cucullo, 2012). As the pore area is generally <20% of the filter area and with a filter being 10–30μm thick, there is little opportunity for contact between the astrocytes and the endothelium as occurs in vivo. Moreover, growing astrocytes on a two-dimensional surface (2D-astrocytes) can affect their phenotype (Maltman & Przyborski, 2010). Recently collagen gels containing different cell types have been used to model the three-dimensional (3D) environment found in tissues in vivo (Lee et al., 2008). Rat astrocytes cultured in 3D matrices are less reactive than 2D-astrocytes (East et al., 2009). They have a different morphology, slower growth rate and fewer stress fibres. 2D-astrocytes spontaneously become reactive (Holley et al., 2005; Wu & Schwartz, 1998) and may secrete inflammatory cytokines and angiogenic factors, which interfere with the maintenance of the endothelial barrier. In addition, astrocytes grown on rigid 2D surfaces are subject to different positional cues to cells in a 3D matrix. For these reasons, 2D-astrocytes do not reflect the phenotype of astrocytes in vivo. In the model developed here, human astrocytes were cultured in a 3D collagen hydrogel, overlaid with a human brain endothelial cell line forming a monolayer on the gel surface. The arrangement allows contact between the endothelium and astrocytes and free movement of larger biomolecules and nanoparticles between the basal surface of the endothelium and the astrocytes in the underlying gel.\n\n\nMaterials and Methods\n\nHuman foetal cortical astrocytes at passage 1, were obtained from ScienCell Research Laboratories (Carlsbad, CA, USA) and were maintained on collagen type-I coated tissue culture dishes in human astrocyte medium (ScienCell Research Laboratories, Carlsbad, CA, USA) including 2% foetal bovine serum and the recommended growth supplements as per the manufacturer. Analysis of these cultures by immunofluorescence indicated that 29.2–46.9% of cells were GFAP+ at the start of the cultures and approximately 90% of the cells were S100β+, indicating that the majority of cells were astrocytes. Fibroblasts (TE7+) constituted 1.7–2.8% of the cells at passages 2–4. Oligodendrocytes (O1+) and microglia (CD68+) were not detectable in the astrocyte cultures.\n\nThe human cerebral microvascular endothelial cell line hCMEC/D3 (Weksler et al., 2005) at passage 24–30 was cultured on collagen-coated flasks or tissue culture inserts in EBM-2 medium (Lonza, Basel, Switzerland) supplemented with 2.5% foetal bovine serum, hydrocortisone, VEGF, epidermal growth factor (EGF), insulin-like growth factor I (IGF-I), human fibroblast growth factor (FGF), ascorbic acid and gentamicin sulphate according to the manufacturer’s formulation. All cells were cultured at 37°C in 100% humid air containing 5% CO2, unless otherwise indicated. The medium was changed every 2–3 days.\n\nCollagen gels containing 0.5 × 106 astrocytes per ml were prepared in 24-well plates, with an initial volume of 450μl cellular collagen gel per well. Gels were composed of a 10% cell suspension of human astrocytes, passage 2–4, in DMEM (Gibco, Thermo Fisher Scientific, UK), 10% 10× minimum essential medium (MEM; Sigma, UK) and 80% type I rat tail collagen (2.5 mg/ml; First Link, Wolverhampton, UK). The collagen was diluted from a 5mg/ml 0.6% acetic acid stock using water, then mixed with MEM and neutralised using sodium hydroxide (assessed by colour change of the phenol red indicator to orange-red), then the mixture was added to the cell suspension and mixed to ensure even distribution of cells before transferring to the 24-well plates or transwell inserts. Gelation took approximately 10 min at 37°C and then astrocyte medium was added. In preliminary experiments the astrocyte density was varied between 0.25 × 106 astrocytes/ml and 2.0 × 106 astrocytes/ml. At higher densities the astrocytes caused the gel matrix to contract and detach from the sides of the wells. Therefore, the optimal density for astrocytes was established as 0.5 × 106 astrocytes/ml for cultures of ≤7 days. If the gels were compressed (see below), or for shorter duration cultures, cell densities up to 1.5 × 106 astrocytes/ml could be used.\n\nFor astrocyte/endothelial co-cultures, astrocyte-containing gels were established as described above, either in 24-well plates or 1cm inserts, containing 0.5 × 106 astrocytes per ml (uncompressed gel) or 1.2 × 106 astrocytes per ml (compressed gel). After gelation astrocytes were cultured for 2h in astrocyte medium. Some of the gels were then stabilised (compressed) using RAFT™ absorbers (TAP Biosystems, Royston, UK – now available from Lonza, UK) for 15 min to remove fluid and reduce gels to less than 10% of their original volume. Uncompressed gels were 1–2mm thick: compressed gels were <0.1mm thick. The gels were maintained for a further 24h in astrocyte medium before being overlaid with hCMEC/D3 cells at a density of 60,000 cells/cm2. The co-cultures were incubated at 37°C for 3 days in EGM2-MV medium, before characterisation or use in transcytosis experiments.\n\nThe details of antibodies used for immunofluorescence and flow cytometry are given in Table 1. 3D cultures were stained by similar methods to conventional 2D cultures, except that the wash times were extended. The gels were rinsed with Hank’s balanced salt solution (HBSS) without Ca++/Mg++, fixed in 4% fresh paraformaldehyde in phosphate buffered saline (PBS) for 45 min and washed for 30 min in three changes of PBS. The gels were then detached from the wells, incubated with 0.05% w/v saponin in PBS and 5% normal goat serum for 1h and washed 3× in PBS before incubation overnight in primary antibody. Gels were washed for 1h with 4 changes of PBS before incubation with secondary antibodies and 1μg/ml Hoechst 33259 for 90 min. Secondary antibodies were 10μg/ml Alexafluor-488 or -555 conjugated goat antibodies to mouse or rabbit IgG (Invitrogen Ltd). Finally the gels were washed for 6h with 12 changes of PBS and imaged using a Leica TCS SP5 confocal microscope (Leica, Germany). From the z-stack of images, a maximal intensity projection image was generated. In the case of 2D cultures, for comparison of treatments, ten images were captured in randomly selected fields using an Olympus BX61 microscope. Fluorescence intensity was analysed using ImagePro software (MediaCybernetics UK, v8) and the percentage of pixels exceeding a threshold pixel-density was measured. The threshold value was set according to the background intensity and this value was constant across all images in each experiment. For analysis of zonula occludens-1 (ZO-1), nuclear and perinuclear staining was excluded from the analysis whereas for claudin-5 (CLDN5), the intensity across the whole cell was measured. In each experiment, the results were normalised to the reference treatment, by dividing the stained area of treated cells by the stained area of control cells. The normalised values from different experiments were then combined to give mean fluorescence intensity ±SEM.\n\n* All mouse primary antibodies are monoclonal. All others are polyclonal.\n\nTo recover cells, the gels were subjected to collagenase digestion. 3D gel cultures of astrocytes were rinsed with HBSS and incubated for 45 min at 37°C with 0.25% collagenase, 10μg/ml DNAse I and 0.147μg/ml TLCK hydrochloride (Tosyl-Lys-chloromethylketone.HCl) in DMEM. Digestion was terminated by addition of 20mM EDTA, and cells recovered by centrifugation at 320g for 5 min. To isolate hCMEC/D3 cells from co-cultures a two-step collagenase digestion was developed. The gels were first treated as above for 15 min and the hCMEC/D3 monolayer detached from the gel surface intact and placed in 0.25% trypsin-EDTA to dissociate the cells (step-1). The collagenase digestion of the gel was continued for a further 30 min in order to recover the astrocytes (step-2).\n\nThe two-step collagenase digest for recovering cell populations was validated by measuring the purity of the astrocyte fraction after co-culture. Endothelial cells (hCMEC/D3) were pre-labelled with a fluorescent tracker, 5-chloromethylfluorescein diacetate (CMFDA), and cultured with the astrocytes for 7 days. The endothelial monolayer was removed and the astrocytes recovered and analysed by flow cytometry (see below). The level of contamination of the astrocytes by endothelium, measured by PECAM-1 staining (endothelial marker) was in the range 5–8%, in different experiments (n=3). Median fluorescence of astrocytes in monoculture in these FACS conditions was 4.7 units, and that of astrocytes isolated from co-cultures was 5.0 units. Isolated endothelium had a median fluorescence of 187 units.\n\nIsolated populations of endothelium or astrocytes were characterised by flow cytometry. Endothelium was labelled with monoclonal antibody to PECAM-1, directly conjugated to PerCP-eFluor-710 for 30 min at 4°C in diluent containing 0.1% NaN3 and 0.1% BSA in PBS. For indirect immunofluorescence, cells were trypsinised, washed and fixed in 4% paraformaldehyde. After two washes, cells were incubated with primary antibodies for 1h at room temperature in PBS containing 0.1% BSA and 2.5% normal goat serum. Control staining was carried out simultaneously with a corresponding matched isotype IgG for mouse primary antibodies, or antibody diluent for rabbit antibodies. Each wash was done in 4ml of PBS; cells were collected by centrifuging at 320g for 5 min. If cell-permeabilization was needed (for primary antibodies recognising intracellular epitopes), saponin (0.05%, final concentration) was added to the antibody diluent and washing buffer. After incubation with primary antibody, the cells were washed twice, then incubated with 10 µg/ml of Alexa Fluor® 488 conjugated secondary antibodies for 45 min. Cells were washed twice, suspended in PBS, then analysed by FACSCalibur™ using Cell Quest software (Becton Dickinson, UK). For each sample, data from 10,000 cells were collected. Each determination was performed with 3 or 4 replicates and the data is expressed as the mean (±SEM) of the median fluorescence from each replicate. Experiments compared different conditions using paired t-tests.\n\n3D co-culture gels prepared on transwell inserts were washed 3× in PBS and fixed in 2.5% glutaraldehyde in Sörensons phosphate buffer (PB) for 1 hour. Post-fixation was carried out with 1% (w/v) osmium tetroxide in 0.1M PB for 1 hour and the inserts were then washed in 0.1M PB for 10 min. The filters were excised from the insert and randomly cut into 2 segments of 3–5mm × 2mm. These segments were progressively dehydrated in a graded series of ethanol from 30% to 100%, embedded in Epon resin (Agar Scientific, UK) and polymerised at 60°C for 48h. Ultrathin sectioning was performed using a Diatome diamond knife on a Leica Ultracut UCT (Leica Biosystems Inc, UK), producing sections of 80–90nm thickness, which were then collected on 2×1mm copper grids coated with pioloform film. The sections were counterstained at room temperature with 4% aqueous uranyl acetate for 35 min, washed 3×, immersed in Reynolds lead citrate, (pH 12), for 10 min in a CO2-free environment, and finally washed 3× before air-drying. The sections were observed on a transmission electron microscope JEM-1400 (Jeol, Japan) operated at an accelerating voltage of 80 kV using a magnification of ×5000 to ×25000. In experiments with gold nanoparticles, silver enhancement (R-Gent SE-EM, Aurion, Netherlands) was carried out for 45 min at room temperature, according to the manufacturer’s protocol, before processing as described above.\n\nFluorescence intensity analysis was carried out using ImagePro software and the percentage of pixels exceeding a threshold pixel-density was measured, with a threshold set at mean+2SD of background. Flow cytometry was carried out using FACSCalibur™ to analyse 104 cells on each determination, using CellQuest software (Becton Dickinson, UK), to give median fluorescence. The gain voltage was set so that 90% of control, stained cells had fluorescence values <10. Cells with fluorescence >10 were considered positive. Graphical representations and statistical analyses were done with Graphpad Prism software, v. 3.0 (Prism, USA). Data were analysed by Anova, followed (if P< 0.05) by either Dunnet’s multiple comparison test, a t-test or a paired t-test as appropriate.\n\n\nResults\n\nThe characteristics of astrocytes cultured in collagen gels (3D-astrocytes) for 7 days were compared to the same cells cultured on flasks (2D-astrocytes). Cells grown on flasks had increased in number approximately 7-fold over this period, equivalent to 2.9 ± 0.08 cell divisions. In contrast, there was no significant increase in the numbers of cells present in the gels after 7 days in culture. The difference between 2D-astrocytes and 3D-astrocytes (P=0.0009, paired t-test, n=4) indicates that culture in the 3D gel environment inhibits astrocyte proliferation. Over the same time period, the percentage of TE7+ fibroblasts increased significantly from 2% to 5% in both 3D-cultures (P=0.0483, paired t-test, n=3) and 2D cultures (P=0.0428, paired t-test, n=3), indicating that fibroblasts can proliferate slowly in the gels.\n\nOver this period the percentage of GFAP+ cells was unchanged in the 3D gels, while the percentage in monolayer cultures fell from 39% to 20% (P=0.0309). The maintenance of GFAP expression in 3D cultures of human astrocytes contrasts with previous results with rat astrocytes, which down-regulate GFAP expression in gel cultures (East et al., 2009). While 90% of the 2D-astrocytes expressed S100β, only approximately half of them were GFAP+. GFAP is often used as a standard marker of astrocytes, but in this study S100β appeared to be more consistently expressed in human astrocytes under different growth conditions than GFAP.\n\nIn the next step, hCMEC/D3 cells were added to the surface of compressed or uncompressed gels containing astrocytes (Figure 1). Compression increased the density of astrocytes and also made the gels considerably more resilient for TEM processing. In order to estimate the volume occupied by the astrocytes in the gels, the total area of the astrocytes was measured as a percentage of the gel, by image analysis of strips across the width of the cultures. In uncompressed gels (1–2mm thick) the astrocytes occupied <1% of the gel, and in compressed gels (40–60μm thick) they occupied 6–10% of the gel (Figure 1). Direct contact between astrocytes at the surface of the gel and endothelium could be seen in TEM (Supplementary Figure 1). However, since the astrocyte density was always <10% of the gel volume the contact area between astrocytes and endothelium was less than 10% of the endothelial monolayer and the permeability of the co-cultures to a 70kDa dextran tracer was not significantly different to monocultures of hCMEC/D3 cells on transwell inserts (P>0.05, t-test, n=3). The upper limit on astrocyte density in the 3D cultures was determined by gel contraction and higher densities of astrocytes could be tolerated in compressed gels, due to their greater rigidity. The model allows analysis of local contacts between the astrocytes and endothelium by fluorescence or electron microscopy, which is an advantage over conventional co-cultures where the endothelium and astrocytes are separated by a filter up to 30µm thick, and direct contact has not been observed.\n\nElectron micrograph of a transverse section (85nm) of a compressed collagen type-1 gel with a co-culture of 3D-astrocytes and hCMEC/D3 endothelial cells; astrocytes occupy 6–10% of the gel as measured by image area. Scale bar = 2μm.\n\nAnother advantage of the gel-based 3D co-culture is that either cell type can be readily observed by microscopy: the transparency of the collagen matrix allows imaging of the endothelial monolayer and astrocytes at different depths in the gel, by conventional light or fluorescence confocal microscopy. Immunofluorescence microscopy was therefore used to examine the expression of characteristic molecules on the endothelium or the astrocytes in the 3D co-cultures. To assess expression of tight junction molecules, endothelial cells were immunostained with anti-ZO-1 and anti-CLDN5. Staining for ZO-1 was continuous around the edge of the cells (Figure 2, upper), whereas CLDN5 was irregular, as described previously (Weksler et al., 2005) in both 2D and 3D cultures. Immunofluorescence staining of the tight junctions on the endothelium indicated that the structural organisation of the junctions was similar to that seen in conventional solo cultures of hCMEC/D3 cells, grown on collagen-coated tissue-culture flasks. Staining of the astrocytes for AQP4 (Figure 2, lower) produced a homogeneous surface staining, with apparently lower expression on astrocytes in 3D co-cultures.\n\nImmunofluorescence staining in situ for expression of ZO-1 in hCMEC/D3 cells (top) and AQP4 in human primary astrocytes (bottom) in 3D gel co-cultures (left) compared with conventional 2D solo cultures on a hard surface (right). 2D and 3D cultures were prepared, cultured, stained and imaged under the same conditions.\n\nTwo previous studies indicated that contact with endothelium can affect expression and localisation of aquaporin-4 (AQP4) in astrocytes (Al Ahmad et al., 2011; Nicchia et al., 2004). To investigate whether co-culture with endothelium was indeed affecting AQP4, we compared expression in 2D-astrocytes, 3D-astrocyte cultures and co-cultures. Culture of astrocytes in 3D gels, by itself causes a reduction in AQP4 expression both by immunofluorescence in situ (by image analysis) and by flow cytometry (Figure 3). The expression was further reduced when the astrocytes were in co-culture with endothelium (Figure 3). Expression of the endothelin receptor, type B (EDNRB) was also reduced by approximately 16%, on 3D-astrocytes by comparison with 2D-astrocytes as measured by flow cytometry (paired t-test, P=0.028 n=3). However, there was no additional change when the 3D-astrocytes were in a co-culture with endothelium.\n\nExpression of AQP4 in 2D-astrocytes, 3D-astrocytes or astrocytes in 3D co-cultures (Co.) measured by immunofluorescence in situ and image analysis (left), or by isolation of the cells and flow cytometry (right). Fluorescence is expressed relative to the median value in 2D-astrocytes. Data points are the mean (±SEM) of median fluorescence values. 3D-astrocytes expressed lower levels of AQP4 than 2D-astrocytes and the expression of 3D-astrocytes further decreased in co-cultures (* P<0.05, paired t-test, n=3).\n\nIt has previously been shown that astrocyte-conditioned medium (ACM) can enhance barrier properties in brain endothelium (Siddharthan et al., 2007) and induce the multi-drug transporter, P-glycoprotein (Megard et al., 2002). We therefore examined whether the factors released from 3D-astrocytes were more or less effective in inducing barrier phenotype in endothelial cells than those from 2D-astrocytes. hCMEC/D3 cells were cultured with 50% conditioned medium from either 2D-astrocytes or 3D-astrocytes and the area of staining measured by image analysis. The ACM from both sources induced ZO-1 and CLDN5 expression in hCMEC/D3 cells to a similar degree (Figure 4), but neither had any effect on P-glycoprotein expression (Figure 4).\n\nExpression of ZO-1, CLDN5 and P-glycoprotein (P-gp) on hCMEC/D3 cells cultured in control medium from endothelium (-), or 50% astrocyte-conditioned medium from either 2D- or 3D-astrocytes. The values obtained by fluorescence microscopy are expressed relative to the median fluorescence of control-treated cells in 3 independent experiments. Data points are the mean (±SEM) of the median fluorescence. ZO-1 and CLDN5 are induced by 2D-astrocyte conditioned medium (* P<0.05, paired t-test, n=3), but there was no significant difference between 2D-astrocytes and 3D-astrocytes.\n\nTo examine the use of the model for assessing nanoparticle transport, 3D co-cultures were set up on transwell inserts on 24-well plates and overlaid with 8μg/ml of 4nm glucose-coated gold nanoparticles (Gromnicova et al., 2013). At intervals of 1–22 hours the cultures were fixed and examined for the localisation of nanoparticles by TEM following silver enhancement (Figure 5, Supplementary Figure 2). The micrographs showed that these nanoparticles were located in the endothelium, the gel matrix and at later time points in astrocytes, suggesting that they move across the endothelium, and then diffuse through the gel matrix and then enter astrocytes (this class of nanoparticle is known to be able to cross plasma membranes directly (Verma et al., 2008)). The model allows quantitation of the numbers of nanoparticles in the endothelium and astrocytes by particle counting, and it allows study of subcellular localisation of the nanoparticles. The model has recently been used for this purpose (Gromnicova et al., 2013). It is also theoretically possible to quantitate cell-associated and matrix-associated gold nanoparticles (by mass spectrometry) following separation of the components of the co-culture by the 2-step collagenase method.\n\nElectron micrograph of a 3D co-culture of human primary astrocytes and hCMEC/D3 cells after application of 8μg/ml glucose-coated gold nanoparticles to the endothelial surface. Nanoparticles (arrows) were detected both in the endothelium and the astrocytes. Scale bar = 500nm.\n\nOne particular advantage of the present 3D gel co-culture is that it allows nanoparticles to be released from the entire basal membrane of the endothelium. In contrast, if endothelium is cultured directly on top of transwell filters, nanoparticles become trapped between the basal plasma membrane and the filter, except where there are pores (Figure 6). Moreover some types of nanoparticle bind strongly to filters and for these nanoparticles transwells are not suitable for quantitation of transfer rates, because of the losses on the filter.\n\nElectron micrograph of a section through hCMEC/D3 cells growing on a transwell filter, 1 hr after application of insulin-coated 4nm gold nanoparticles to the apical surface. The cells were fixed and nanoparticles visualised by silver enhancement. Nanoparticles (arrows) are trapped between the filter and basal membrane, except where there are pores (P) in the filter. Scale bar = 1µm.\n\nWe also examined the model to determine whether it could be used to assess leukocyte migration across the endothelium and through the gel. Collagen hydrogel co-cultures were established in transwell inserts and the endothelium and astrocytes were activated by treatment with 1–10ng/ml TNFα plus IFNγ, either in the upper or lower chambers. Earlier studies have established that these conditions induce appropriate adhesion molecules and chemokine synthesis by the endothelium (Subileau et al., 2009). The endothelium was then overlaid with 105 Jurkat cells labelled with CMFDA and incubated for 16–24h. At the end of the incubation the cultures were observed by fluorescence microscopy and TEM. Confocal microscopy identified fluorescent lymphocytes associated with the endothelial monolayer, and with smaller numbers up to 300μm deep in the gel (Supplementary Figure 3). Migration into the gel was only seen with uncompressed gels. The TEM pictures demonstrated that the lymphocytes had migrated through the endothelium, exhibiting the classical appearance of emperipolesis (Figure 7). The micrographs indicated that the majority of the leukocytes detected had accumulated below the basal plasma membrane of the endothelium but had not migrated into the gel at 24h (Supplementary Figure 4). Accumulation of leukocytes below the plasma membrane of brain endothelium but within the glia limitans (perivascular cuffing) is also a characteristic of leukocyte migration into the CNS in vivo.\n\nMigration of Jurkat cells in a 3D-astrocyte-endothelial co-culture. a) At 3h Jurkat cells (J) are seen above and below the endothelial cells (E), with some cellular processes interleaved in the endothelial monolayer. b) At 24h the Jurkat cells are located between the endothelium and the collagen gel. The arrow indicates an intact inter-endothelial junction. Transendothelial migration occurs by emperipolesis. Scale bars = 1μm.\n\n\nDiscussion\n\nThe 3D co-culture model was developed to assess interactions between human brain endothelium and astrocytes and for its potential to measure trans-endothelial migration of nanoparticles and leukocytes. The configuration of these cultures offers three potential advantages: firstly astrocytes can directly contact the endothelium; secondly, compared to 2D hard surface cultures, the 3D soft collagen gel matrix provides a physiologically more realistic cell environment, as indicated by inhibition of astrocyte proliferation; thirdly, nanoparticles, cells or biological agents released from the basal plasma membrane of the endothelium can directly enter the gel and/or astrocytes.\n\nThe model also has great potential for investigating the phenotype of the two cell types in co-culture, by light and fluorescence microscopy due to the transparency of the matrix. Compressed gels are also readily processed for TEM. Moreover as both cell types can be recovered separately by collagenase digestion, they can be individually analysed by many other techniques.\n\nThe second major use of this model was intended to be in measuring the movement of nanoparticles, across brain endothelium and into astrocytes. Delivery of therapeutic transgenes to cells within the CNS holds great potential for treatment of CNS diseases (Manfredson & Mandel, 2010), but developing systems that deliver the transgene to the target cell within the CNS is a challenge. Nanoparticles have the capacity to act both as drug carriers and transporters of therapeutic transgenes (Kanwar et al., 2012). However there is currently no convenient system for measuring trans-endothelial movement of the nanoparticles in vitro and localising them in target cells. This model is well suited for this purpose. Nanoparticles can be tracked and quantitated by TEM (or light microscopy for larger particles). We have now used this model to quantitate delivery of different classes of nanoparticle to astrocytes in the gels and determine the rate and route of transfer across the endothelium (Gromnicova et al., 2013). We also evaluated the model for leukocyte migration. In comparison with another recently-developed astrocyte/endothelial co-culture (Takeshita et al., 2014), specifically designed for measuring leukocyte migration, our model lacks shear stress, and is less suited for quantitation of leukocyte traffic, but can be used for characterising the trans-migrating cells and identifying their routes of migration.\n\nAnother related model which includes rat brain endothelium (RBE4-line), rat primary astrocytes and primary pericytes, in a collagen gel has been developed previously (Al Ahmad et al., 2011). In this case all cells were incorporated into the gel matrix as the model was designed to investigate angiogenesis, and tubulogenesis. In comparison, it is less well suited for the analysis of mechanisms of transcytosis.\n\n\nConclusions\n\nThis 3D co-culture system with quiescent astrocytes in a collagen hydrogel overlaid with a monolayer of brain endothelium can be used for examining astrocyte-endothelial interactions; the two cell types can directly contact each other, although the overall contact area is limited. The model can also be used for investigating leukocyte migration across brain endothelium. It is particularly well suited for measuring the delivery of nanoparticles, transgenes and larger biological molecules to cells of the CNS, following transfer across the endothelium, because these larger entities can move freely from the basal surface of the endothelium, through the gel matrix and into the target cells.\n\n\nData availability\n\nF1000Research: Dataset 1. Raw data for the statements made in the Results ‘Phenotype of human astrocytes in 3D gels’, 10.5256/f1000research.7142.d106539\n\nF1000Research: Dataset 2. Raw data for aquaporin-4 expression (Figure 3), 10.5256/f1000research.7142.d106541\n\nF1000Research: Dataset 3. Raw data for expression of ZO-1, CLDN5 and Pgp (Figure 4), 10.5256/f1000research.7142.d106543", "appendix": "Author contributions\n\n\n\nPS and DM conceived the study, designed the experiments and drafted the manuscript. PS and RG carried out the experiments. HD and JP provided guidance on electron microscopy and development of the 3D cultures, respectively. IR assisted with study design and data analysis. 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\nDM is on the scientific advisory board of Midatech Pharma plc.\n\n\nGrant information\n\nThis work was supported by the BBSRC (Ignacio Romero, Project BB/K009184/1) and a grant to David Male from the Migraine Trust (Project 2007/04). Midatech Ltd kindly supplied the nanoparticles and provided support for Radka Gromnicova.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe are most grateful to Frances Colyer for assistance with electron microscopy and to Dongsheng Wu for assistance with leukocyte migration assays.\n\n\nSupplementary material\n\nSupplementary Figures 1–4.\n\nSupplementary Figure 1: Electron micrograph of a 3D-astrocyte endothelial cell co-culture (uncompressed). Astrocytes constitute approximately 1% of the gel volume and in some places are in direct contact with the basal plasma membrane of the hCMEC/D3 cells. Scale bar = 2mm. Supplementary Figure 2: Electron micrograph of hCMEC/D3 cell 8 hrs after application of glucose-coated 4nm gold nanoparticles to the apical surface. The cells were fixed and nanoparticles visualised by silver enhancement. Nanoparticles are present in the cytosol and below the basal membrane (arrows). Scale bar = 500nm. Supplementary Figure 3: Confocal micrograph of CMFDA-labelled Jurkat cells migrating into a 3D uncompressed co-culture. The endothelium was stimulated with 1ng/ml TNFa and 1ng/ml IFNg at time 0. Jurkat cells were applied to the apical surface and allowed to migrate for 24h before fixation. Nuclei of the endothelial cells are labelled with DAPI. Some Jurkat cells are associated with the endothelial monolayer, while others have migrated up to 300mm into the gel. Scale bar = 200mm. Supplementary Figure 4: Electron micrograph of Jurkat cells (J) migrating into a 3D uncompressed co-culture. Migration into the gel occurs at a point where the endothelial monolayer (E) is incomplete. Some astrocytes (A) are also visible in this section. Scale bar = 1mm.\n\nClick here to access the data.\n\n\nReferences\n\nAbbott NJ, Patabendige AA, Dolman DE, et al.: Structure and function of the blood-brain barrier. Neurobiol Dis. 2010; 37(1): 13–25. PubMed Abstract \| Publisher Full Text\n\nAl Ahmad A, Taboada CB, Gassmann M, et al.: Astrocytes and pericytes differentially modulate blood-brain barrier characteristics during development and hypoxic insult. J Cereb Blood Flow Metab. 2011; 31(2): 693–705. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nButt AM, Jones HC, Abbott NJ: Electrical resistance across the blood-brain barrier in anaesthetized rats: a developmental study. J Physiol. 1990; 429(1): 47–62. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSreekanthreddy P, Gromnicova R, Davies H, et al.: Dataset 1 in: A three-dimensional model of the human blood-brain barrier to analyse the transport of nanoparticles and astrocyte/endothelial interactions. F1000Research. 2015a. Data Source\n\nSreekanthreddy P, Gromnicova R, Davies H, et al.: Dataset 2 in: A three-dimensional model of the human blood-brain barrier to analyse the transport of nanoparticles and astrocyte/endothelial interactions. F1000Research. 2015b. Data Source\n\nSreekanthreddy P, Gromnicova R, Davies H, et al.: Dataset 3 in: A three-dimensional model of the human blood-brain barrier to analyse the transport of nanoparticles and astrocyte/endothelial interactions. F1000Research. 2015c. Data Source\n\nSubileau EA, Rezaie P, Davies HA, et al.: Expression of chemokines and their receptors by human brain endothelium: implications for multiple sclerosis. J Neuropathol Exp Neurol. 2009; 68(3): 227–240. PubMed Abstract \| Publisher Full Text\n\nTakeshita Y, Obermeier B, Cotleur A, et al.: An in vitro blood-brain barrier model combining shear stress and endothelial cell/astrocyte co-culture. J Neurosci Methods. 2014; 232: 165–172. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVerma A, Uzun O, Hu Y, et al.: Surface-structure-regulated cell-membrane penetration by monolayer-protected nanoparticles. Nat Mater. 2008; 7(7): 588–595. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWeksler BB, Subileau EA, Perrière N, et al.: Blood-brain barrier-specific properties of a human adult brain endothelial cell line. FASEB J. 2005; 19(13): 1872–1874. PubMed Abstract \| Publisher Full Text\n\nWeksler B, Romero IA, Couraud PO: The hCMEC/D3 cell line as a model of the human blood brain barrier. Fluids Barriers CNS. 2013; 10(1): 16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWolburg H, Lippoldt A: Tight junctions of the blood-brain barrier: development, composition and regulation. Vascul Pharmacol. 2002; 38(6): 323–337. PubMed Abstract \| Publisher Full Text\n\nWu VW, Schwartz JP: Cell culture models for reactive gliosis: new perspectives. J Neurosci Res. 1998; 51(6): 675–681. PubMed Abstract \| Publisher Full Text" }	[ { "id": "11397", "date": "16 Dec 2015", "name": "Abraham J. Al Ahmad", "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 sounds technically good and the results were innovative. However, I have some recommendation or comments that would make bring this study to its highest potential.Figure 2: A Claudin-5 immunostaining would be appreciated, especially to observe how the presence of astrocytes may improve claudin-5 immunoreactivity in hCMEC/D3 cells. What happens to adherent junctions such as VE-cadherin, beta-catenin or PECAM-1? Having such immunocytochemistry data in addition would greatly help.A GFAP staining would be appreciated. Figure 4: I have some concerns about the absence of error bars in P-gp control group, although CLDN5 and ZO1 control groups display one. Please keep it consistent. Figure 7: A true negative control such as TE7 fibroblasts is needed.", "responses": [ { "c_id": "1761", "date": "21 Jan 2016", "name": "David Male", "role": "Author Response", "response": "1. We have previously shown that Claudin-5 immunostaining on hCMEC-D3 cells is more patchy than ZO-1 staining (Weksler et al, 2005). This may partly account for the relatively low trans-endothelial resistance of this cell line. Cnsequently claudin-5 staining was not done. Data in figure 4 suggests that claudin-5 is induced by astrocytic factors although there was no significant difference between 2D and 3D culture factors. PECAM was unchanged in solo-culture and co-culture (data not presented) and VE-cadherin was not investigated. A GFAP stain has been added to supplementary material.2. In figure 4 Pgp was measured by flow cytometry with data adjusted to the untreated cells on each experiment (=100%). For this reason there are no error bars on the untreated cells. For complete understanding of how the data was normalized see dataset 4.3. A negative control image has been added to the supplementary material." } ] }, { "id": "11245", "date": "21 Dec 2015", "name": "Christopher C.W. Hughes", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThere is a tremendous demand for a reproducible and representative model of the human blood brain barrier and many versions have been tried. This paper represents an interesting variation, especially in the dispensing of transwells, which impose an unacceptable separation between the endothelial cells and the astrocytes. There are a few issues I would like to see addressed, either by experimentation or additional discussion: Pericytes are also an important component of the microvasculature and their addition here would likely strengthen the relevance of the model. Given the importance of astrocytes in promoting development of the BBB it would be interesting to know whether they modulate additional markers of the BBB, such as ABC transporters, other than ZO-1 and Claudin 5 (both of which are only weakly induced). The failure to induce pgp and the relatively high leakiness of the monolayer are concerns as pgp expression and a tight barrier are both key features of the BBB in vivo. Was a non-binding antibody used for the negative control in IF experiments? If so, this should be stated. AQP4 is reduced in overall expression in 3D culture, but is it relocalized to the contact points between the astrocytes and the EC. This could be checked by confocal microscopy. In Fig. 6, the arrows are missing. In Fig. 7, “J” and “E” are missing from the figures.", "responses": [ { "c_id": "1760", "date": "21 Jan 2016", "name": "David Male", "role": "Author Response", "response": "1. We agree with the reviewer that incorporating pericytes will make the model more relevant. Some previous studies (Al Ahmad, 2010) have included the pericytes in 3D collagen gels. It would be closer to the in vivo conditions if we could grow a layer of pericytes between the endothelial monolayer and 3D cultured astrocytes. However, this will increase the complexity of the model, so to keep the model simple and suitable to study transcytosis, we decided to include only two cell types - the brain endothelium which must be crossed by nanoparticles and one example of a target cell, the astrocytes. 2. ABC transporter B1 also known as P-gp-1 is expressed in hCMEC/D3 at similar levels to primary brain endothelium and was found not to be regulated by either 2D cultured or 3D cultures astrocytes. These results are presented in figure 4.3. Although it is highly desirable that in vitro modesl should simulate all aspects of the blood brain barrier in vivo, this has not been possible with any models produced to date. In this case we make it clear where the model is less useful (tight barrier properties and permeability for small molecules) but it is nevertheless useful for the functions indicated - nanoparticle transcytosis and targeting to astrocytes and leukocyte transendothelial migration.4. Immunofluorescence : For mouse monoclonal antibodies, the corresponding isotype immunoglobulin was used as control whereas for rabbit polyclonal antibodies only antibody diluent was used as control. This has been consistently followed in this study because we found that normal rabbit IgG produced a weak non-specific binding, increasing the image backgrounds.5. We agree with the referee and as mentioned in the main text, the model allows analysis of local contacts between the astrocytes and endothelium by fluorescence or electron microscopy, which is an advantage over conventional co-cultures. However, since the astrocyte density was always <10% of the gel volume the contact area between astrocytes and endothelium was less than 10% of the endothelial monolayer, number of contacts were less. Within these limited observations, we did not see relocalization of the AQP4 to the contacting end feet of astrocyte.6/7 Additional annotations made to figures." } ] } ]	1	https://f1000research.com/articles/4-1279
https://f1000research.com/articles/5-90/v1	21 Jan 16	{ "type": "Case Report", "title": "Case Report: Bronchial associated lymphoid tissue lymphoma and Mycobacterium chelonae", "authors": [ "Joana Neves", "Pedro Ferreira", "Gilberto Silva", "Lília Andrade", "Pedro Ferreira", "Gilberto Silva", "Lília Andrade" ], "abstract": "Bronchial-associated lymphoid tissue (BALT) lymphoma is a rare condition that accounts for only 0.5-1% of all malignant lung tumours. We present the case of a 66-year-old man admitted with pneumonia for further study and therapy. Initially the sputum was positive for Mycobacterium tuberculosis complex using polymerase chain reaction technology and antituberculous therapy was initiated. Due to the lack of imagiological improvement, the patient underwent a pulmonary transthoracic biopsy that revealed BALT lymphoma. Months later, Mycobacterium chelonae was identified and specific therapy was started with clarithromycin and tobramycin, before initiating BALT treatment with cyclophosphamide. There are only a few documented cases of BALT lymphoma associated with Mycobacterium. In this case M. chelonae might have been present before BALT lymphoma, contributing as an immunologic stimulus, or appeared afterwards, in the neoplastic context. BALT has an indolent evolution with a good prognosis and that is the reason why some experts favour a “watchful waiting” option.", "keywords": [ "BALT lymphoma", "Mycobacterium chelonae", "diagnosis", "treatment" ], "content": "Introduction\n\nExtranodal marginal zone B cell lymphoma (MZL) is a low grade B cell lymphoma of mucosa associated lymphoid tissue (MALT). Primary pulmonary MALT, also called bronchial-associated lymphoid tissue (BALT), accounts for 0.5–1% of all malignant lung tumours and about 90% of all lung lymphomas1,2. BALT represents 15% of all MALT lymphomas1,3.\n\nBALT lymphoma has been associated with chronic antigenic stimulation by autoimmune disease, smoking, infection or chronic inflammation, but a well-established connection has not yet been described3–5. The paradigmatic example of that antigenic stimuli is the development of gastric MALT in Helicobacter pylori infection with chronic gastritis1. There are some reports in literature correlating Mycobacterium avium complex and Mycobacterium tuberculosis infection with BALT lymphoma5–8.\n\nNontuberculous mycobacterium (NTM) are ubiquitous in the environment, frequently colonize the skin, digestive and respiratory tract, sometimes developing disease, mainly in immunosuppressed chronic diseases patient’s9,10. Mycobacterium chelonae is a rapidly growing mycobacteria that has been implicated as an infrequent lung pathogen. Patients with severe underlying structural lung disease such as cystic fibrosis and bronchiectasis are the most predisposed11.\n\n\nCase report\n\nWe present a 66-year-old man’s case, with subfebrile temperature, two months’ history of persistent cough, purulent sputum, occasional small haemoptysis, anorexia and non-quantified weight loss. He reported no clinical response to previous antibiotherapy with amoxicillin (500 mg orally 3 times a day for 7 days) and after moxifloxacin (400 mg orally once a day for 7 days). The patient reported a gastric lymphoma at the age of 40, treated with chemotherapy for which we have no detailed information. He had a 32-pack-year tobacco smoking history. He had no changes on physical examination. Initial chest radiography showed a bilateral heterogeneous opacity, covering the lower two thirds of the left hemithorax and the lower third of the right hemithorax (Figure 1). Inflammatory parameters, such as eritrocit sedimentation rate and C-reactive protein were normal as well as blood gas. Pneumonia was considered and antibiotherapy with amoxicillin/clavulanic acid (1000 mg/200 mg intravenously every 8 hours) and clarithromycin (500 mg orally twice a day) was initiated, which held during seven days. Later chest CT showed multiple foci of parenchymal consolidation with air bronchogram on the left upper lobe, lingula, apical segment of the left lower lobe, posterior segment of the right upper lobe and apical right lower lobe (Figure 2). Mantoux test was negative. He had elevated IgM (1451 mg/dl) as well as negative serology for HIV, B and C hepatitis. No endoscopic abnormalities were found on flexible bronchoscopy. Gastric endoscopy showed an antral gastritis with negative Helicobacter pylori biopsy test. Bacteriologic analysis of three sputum specimens were negative for either aerobic and anaerobic bacteria or acid fast bacilli, but positive for Mycobacterium tuberculosis complex (MTC) using polymerase chain reaction (PCR) technology, in two out of three samples, bronchial aspirate was bacteriologically negative. The patient was initiated on antituberculous treatment (ATT) with isoniazid (300 mg/day), rifampicin (600 mg/day), pyrazinamide (1500 mg/day) and ethambutol (1200 mg/day), orally, and was forwarded to an outpatient centre for respiratory diseases. Six weeks later, Lowenstein-Jensen cultures showed the presence of NTM. ATT was maintained due to clinical improvement while waiting for NTM identification. The lack of imagiologic improvement in the meantime, led to transthoracic lung biopsy realization. Immunohistochemistry showed positive lymphocyte identification to CD20 and BCL2, and CD23, CD10, CD5 and a negative CD3, consistent with the diagnostic of BALT lymphoma. There was no evidence of other extranodal or nodal involvement in complementary imagiological study. Haematologists decided to revaluate the patient’s condition after the end of ATT, taking into account his clinical stability. In the meantime, NTM was identified as M. chelonae and the laboratory informed us that the initial positive PCR for MTC was a laboratorial contamination. The drug resistance patterns were not performed. At this time new sputum samples were bacteriologically negative and ATT was stopped, with six months of treatment.\n\nChest radiography: bilateral heterogeneous opacity, covering lower two thirds of the left hemithorax and the lower third of the right hemithorax (A: posteroanterior view; B: lateral right view).\n\nThoracic computed tomography, axial view: pulmonary consolidation with air brochogram, in the left upper lob, lingual, apical segment of the left lower lobe, posterior segment of the right upper lobe and apical right lower lobe (A: pulmonary window; B: with contrast).\n\nOne month later the patient resumed his previous respiratory symptoms. Specific therapy for M. Chelonae with tobramycin (150 mg intramuscularly once a day) and clarithromycin (500 mg orally twice a day) was initiated, but discontinued due to nephro and hepatic toxicity, after seven weeks of treatment. Haematologists at this time decided to begin treatment with cyclophosphamide (50 mg orally once a day). After 6 months of cyclophosphamide treatment the patient showed significant clinical improvement, without any signs of pulmonary infection and partial imagiologic resolution. For this reason the patient remains in treatment, waiting for a revaluation with new clinical and imagiologic data.\n\n\nDiscussion\n\nThe diagnosis of BALT lymphoma is challenging and frequently misdiagnosed as pneumonia, pulmonary tuberculosis or interstitial lung disease, because clinical and radiologic findings are nonspecific. According to retrospective analysis, average time to achieve diagnosis is around 20 months4. Chronic cough, sputum, progressive dyspnoea, fatigability, fever, night sweats and weight loss are the most common manifestations. Imagiologic findings are generally nonspecific, such as single or multiple nodules, consolidation areas, bronchiectasis, bronchiolitis phenomena or diffuse interstitial lung disease2,4,12.\n\nPulmonary tuberculosis was assumed initially with the identification of Mycobacterium tuberculosis complex by PCR, leading us to treat the patient with ATT. When the presence of an NTM was identified in two out of three sputum specimens together with clinical and imagiologic changes, an infection was assumed according to the criteria of American Thoracic Society (ATS) for NTM lung disease12. Nevertheless the doubt about M. chelonae infection or colonization emerged, when BALT lymphoma was diagnosed, because imagiologic changes could either be attributed to BALT or M. chelonae, which compromises ATS criteria for infection. Also the absence of clinical signs of pulmonary infection after stopping a short period of specific therapy, but with the beginning of chemotherapy, led us to consider that M. chelonae was possibly just a colonizer.\n\nWe were unable to establish if M. chelonae had a role as a chronic antigenic stimulus to BALT lymphoma or if it was BALT lymphoma that led to secondary infection or colonization by M. chelonae.\n\nOptimal therapy is unknown. Chemotherapy with CHOP or R-CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone/prednisolone, with or without rituximab) is the most common treatment described by authors; however monotherapy with cyclophosphamide also presents a high rate of disease control and can be used as a single agent13,14. Some experts favour a “watchful waiting” option in early stages and on asymptomatic patients due to the indolent evolution and the good prognosis of BALT, generally expected to have more than 80–90% of a five year survival rate14,15.\n\nIn our case a less aggressive approach was decided due to the indolent evolution of BALT, the clinical stability of the patient, and to avoid a severe immunocompromised state, which could lead to a mycobacterial infection.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient.", "appendix": "Author contributions\n\n\n\nJN and LA conceived the study and carried out the research. All authors contribute in the diagnosis and follow up of the patient. JN 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\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nBorie R, Wislez M, Thabut G, et al.: Clinical characteristics and prognostic factors of pulmonary MALT lymphoma. Eur Respir J. 2009; 34(6): 1408–16. PubMed Abstract \| Publisher Full Text\n\nNoguchi S, Yatera K, Kido T, et al.: Pulmonary mucosa-associated lymphoid tissue (MALT) lymphoma with multiple thin-walled pulmonary cysts: a case report and review of the literature. Intern Med. 2013; 52(20): 2325–2329. PubMed Abstract \| Publisher Full Text\n\nZinzani PL, Polleti V, Zompatori M, et al.: Bronchus-associated lymphoid tissue lymphomas: an update of a rare extranodal maltoma. Clin Lymphoma Myeloma. 2007; 7(9): 566–572. PubMed Abstract \| Publisher Full Text\n\nHuang H, Lu ZW, Jiang CG, et al.: Clinical and prognostic characteristics of pulmonary mucosa-associated lymphoid tissue lymphoma: a retrospective analysis of 23 cases in a Chinese population. Chin Med J (Engl). 2011; 124(7): 1026–30. PubMed Abstract\n\nKlein TO, Soll BA, Issel BF, et al.: Bronchus-associated lymphoid tissue lymphoma and Mycobacterium tuberculosis infection: an unusual case and a review of the literature. Respir Care. 2007; 52(6): 755–8. PubMed Abstract\n\nVadhiraja BM, Srinivasan R, Fernandes DJ, et al.: Bronchial associated lymphoid tissue lymphoma with bronchiectasis in a pretreated tuberculosis patient. Chest. 2005; 15(2): 225–227. Publisher Full Text\n\nGaur S, Trayner E, Aish L, et al.: Bronchus-associated lymphoid tissue lymphoma arising in a patient with bronchiectasis and chronic Mycobacterium avium infection. Am J Hematol. 2004; 77(1): 22–5. PubMed Abstract \| Publisher Full Text\n\nInadome Y, Ikezawa T, Oyasu R, et al.: Malignant lymphoma of bronchus-associated lymphoid tissue (BALT) coexistent with pulmonary tuberculosis. Pathol Int. 2001; 51(10): 807–11. PubMed Abstract \| Publisher Full Text\n\nPrimm TP, Lucero CA, Falkinham JO 3rd: Health impacts of environmental mycobacteria. Clin Microbiol Rev. 2004; 17(1): 98–106. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKothavade RJ, Dhurat RS, Mishra SN, et al.: Clinical and laboratory aspects of the diagnosis and management of cutaneous and subcutaneous infections caused by rapidly growing mycobacteria. Eur J Clin Microbiol Infect Dis. 2013; 32(2): 161–88. PubMed Abstract \| Publisher Full Text\n\nHsieh HC, Lu PL, Chen TC, et al.: Mycobacterium chelonae empyema in an immunocompetent patient. J Med Microbiol. 2008; 57(Pt 5): 664–7. PubMed Abstract \| Publisher Full Text\n\nGriffith DE, Aksamit T, Brown-Elliott BA, et al.: An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. Am J Respir Crit Care Med. 2007; 175(4): 367–416. PubMed Abstract \| Publisher Full Text\n\nJoshi M, Sheikh H, Abbi K, et al.: Marginal zone lymphoma: old, new, targeted, and epigenetic therapies. Ther Adv Hematol. 2012; 3(5): 275–290. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang B, Zhang C, Wang B, et al.: Comparisons of Surgery and/or Chemotherapy in the Treatment of Primary Pulmonary Mucosa-Associated Lymphoid Tissue Lymphoma. Ann Thorac Cardiovasc Surg. 2015; 21(2): 109–13. PubMed Abstract \| Publisher Full Text\n\nTroch M, Streubel B, Petkov V, et al.: Does MALT lymphoma of the lung require immediate treatment? An analysis of 11 untreated cases with long-term follow-up. Anticancer Res. 2007; 27(5B): 3633–3637. PubMed Abstract" }	[ { "id": "12305", "date": "18 Feb 2016", "name": "Chunxue Bai", "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\nJoana Neves et al. presented a case of BALT lymphoma. This patient was admitted with pneumonia and then his sputum was positive for Mycobacterium tuberculosis complex using polymerase chain reaction technology. However, there was no imagiological improvement after antituberculous therapy. Therefore, the patient underwent a pulmonary transthoracic biopsy that revealed BALT lymphoma. After 6 months of cyclophosphamide treatment the patient showed significant clinical improvement. Please provide pertinent laboratory data at the time of the patient’s initial evaluation, such as white blood cell count, erythrocyte sedimentation rate, antinuclear antibody titer, β2-microglobulin. Please provide relevant computed tomography findings following antituberculous treatment and cyclophosphamide treatment. Please provide microscopic examination results about pulmonary transthoracic biopsy.", "responses": [] }, { "id": "15259", "date": "08 Aug 2016", "name": "Jose C. Villasboas Bisneto", "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 the case of a 66-year-old man presenting with subacute respiratory and constitutional symptoms. Interestingly the patient has a history of gastric lymphoma but little additional information is available for this very pertinent piece of his medical history. Following failure of empiric therapy for typical pulmonary pathogens and a misleading polymerase chain reaction (PCR) result showing Mycobacterium tuberculosis complex (MTC) in the sputum (later found to be laboratorial contamination) the diagnosis of Mycobacterium chelonae infection is made. This was based on sputum cultures that eventually resulted positive for nontuberculous mycobacterium (NTM) six weeks after incubation. It is important to note that the authors mention that antituberculous therapy (ATT) was continued despite the results since the patient was improving clinically.\n\nDue to lack of radiological improvement a transbronchial biopsy was performed revealing a lymphocytic infiltrate positive for CD20, BCL2, CD23, CD10, CD5 and negative for CD3. This serves as the basis for the diagnosis of bronchial-associated lymphoid tissue (BALT) lymphoma in this patient. In my opinion this is the weakest link in the logical chain for this case report. No evidence of clonality was demonstrated by analysis of surface immunoglobulin light chain or IgH gene rearrangement. Testing for chromosomal translocations typically associated with mucosa-associated lymphoid tissue (MALT) lymphomas such as t(11;18)q21;q21) or t(11;14)(p22;q32) was not performed. Although the absence of such translocations does not necessarily exclude MALT lymphomas, a positive result would substantiate the claimed diagnosis. As demonstrated by Ye and colleagues1, BALT lymphomas harbor the t(11;18) translocation in 38.3% of cases. Another useful test would be BCL10 staining, knowing that 26% of t(11;18)-negative cases will have moderate BCL10 expression based on results from the same cohort. In the absence of these additional tests the authors leave their claim for the diagnosis of BALT lymphoma exceedingly vulnerable to challenge. A reactive lymphoid infiltrate therefore cannot be excluded as a reasonable alternative diagnosis.\n\nThe report progresses to describe that the patient was treated with oral cyclophosphamide (CTX) resulting in clinical and radiological improvement. The association between treatment with CTX and improvement however can only offer circumstantial evidence to support that BALT lymphoma was indeed the correct diagnosis. One could argue that response to specific therapy against M. chelonae was delayed and coincided with the initiation of therapy with CTX. Alternatively, one could also argue that the correct diagnosis in this patient is interstitial lymphocytic pneumonia (ILP) for which immunosuppressive agents such as CTX has demonstrated efficacy.\n\nThe discussion is naturally built on the assumption that BALT lymphoma is the correct diagnosis in this case. Since this publication is largely targeted to the general audience I would suggest including a list of differential diagnosis for patients with suspected BALT lymphomas (preferably in tabular format). Another point to add in the discussion is the role of single-agent rituximab or radiotherapy in the treatment of patients with early-stage disease.\n\nIn summary, given that the central hypothesis of this manuscript (diagnosis of BALT lymphoma) is supported by weak evidence I would suggest the following revisions before the manuscript can be considered for publication:\n\nProvide additional evidence for the diagnosis of BALT lymphoma by demonstrating clonality (minimum) and/or specific genetic lesions associated with MALT lymphomas.\n\nConsider another attempt obtaining more information about this patient’s previous history of gastric lymphoma. Should you be able to demonstrate that this patient had MALT lymphoma of the stomach this would open another set of questions such as: is the BALT lymphoma truly a primary event or is it a recurrence from the previously treated MALT lymphoma? Does this patient carry a genetic predisposition for this disease?\n\nConsider a list of differential diagnosis to be considered in someone suspected to have BALT lymphoma, possibly in tabular format for ease of read.\n\nAdd to the discussion the role of single-agent rituximab or radiotherapy in early-stage disease.\n\nI would be happy to review the revised manuscript after changes are made.", "responses": [] } ]	1	https://f1000research.com/articles/5-90
https://f1000research.com/articles/5-89/v1	20 Jan 16	{ "type": "Review", "title": "Telomeres and telomerase as therapeutic targets to prevent and treat age-related diseases", "authors": [ "Christian Bär", "Maria A. Blasco", "Christian Bär" ], "abstract": "Telomeres, the protective ends of linear chromosomes, shorten throughout an individual’s lifetime. Telomere shortening is a hallmark of molecular aging and is associated with premature appearance of diseases associated with aging. Here, we discuss the role of telomere shortening as a direct cause for aging and age-related diseases. In particular, we draw attention to the fact that telomere length influences longevity. Furthermore, we discuss intrinsic and environmental factors that can impact on human telomere erosion. Finally, we highlight recent advances in telomerase-based therapeutic strategies for the treatment of diseases associated with extremely short telomeres owing to mutations in telomerase, as well as age-related diseases, and ultimately aging itself.", "keywords": [ "Telomeres", "Telomerase", "Aging", "telomere length", "short telomeres" ], "content": "Telomere structure, function and maintenance\n\nTelomeres are heterochromatic structures located at the ends of linear chromosomes formed by DNA tandem repeats bound by specialized protein complexes, which exert a protective function. A proper telomere structure prevents chromosome ends from being recognized as DNA strand breaks, thus preventing illegitimate homologous recombination between telomeres as well as chromosome end-to-end fusions1. In vertebrates, telomeric DNA is composed of up to thousands of TTAGGG hexanucleotide repeats that are bound by a six-protein complex known as shelterin, which encompasses TRF1, TRF2, POT1, TIN2, TPP1, and RAP12. TRF1 and TRF2 directly bind double-stranded telomeric repeats, whereas POT1 recognizes the single-stranded telomeric G-rich 3’ overhang. TIN2 binds to TRF1 and TRF2 through distinct domains and also recruits a TPP1-POT1 heterodimer, thus bridging different shelterins to organize the telomere cap2. Finally, RAP1 is recruited to telomeres by TRF2, but can also bind throughout chromosome arms to regulate transcription, playing an important role in protection from obesity and metabolic syndrome in mice3–5. Interestingly, all shelterins except RAP1 are essential for life6–8, owing to the fact that RAP1 is the only shelterin dispensable for telomere protection3,9,10.\n\nTelomeres are proposed to be further stabilized by the formation of a protective T-loop lariat structure. The single-stranded 3’ overhang loops back and invades double-stranded telomeric DNA in a TRF2-dependent manner11,12. Thus, the T-loop sequesters the ends of chromosomes and provides a mechanism to prevent the full activation of a DNA damage response typically observed at most types of DNA ends13.\n\nImportantly, owing in part to the so-called “end replication problem”, telomeres shorten during each cell duplication cycle due to the inability of replicative DNA polymerases to fully replicate the 3’ ends of linear chromosomes14,15. In particular, the removal of RNA primers, which provide the required 3’OH group for addition of dNTPs by DNA polymerases, renders the newly synthesized DNA strand shorter than the parental template. Thus, chromosomes progressively shorten from both ends upon repeated cell division, a process which in the context of the organism contributes to progressive telomere shortening with aging in all cell types where it has been studied16. When telomeres reach a critically short length they are detected by the DNA repair systems as DNA damage and elicit cell cycle arrest and cell death responses17. Thus, telomere shortening underlies the “molecular clock” proposed by Hayflick to explain the limited lifespan of cells in culture, or “Hayflick limit”17,18.\n\nTelomerase is a DNA reverse transcriptase polymerase (telomerase reverse transcriptase [TERT]) which uses an RNA template (telomerase RNA component [TERC]) for de novo addition of telomeric DNA onto telomeres, thus compensating for the telomere erosion caused by cell divisions19. Indeed, overexpression of telomerase is sufficient to counteract telomere attrition and to indefinitely extend the replicative lifespan of primary cells in culture in the absence of genomic instability, transforming them into cancerous cells20–22. However, high telomerase expression is normally restricted to early stages of embryonic development (i.e. the blastocyst stage in mice and humans) and to pluripotent embryonic stem cells23,24. Thus, adult mammalian tissues including adult stem cell compartments do not express sufficient amounts of telomerase to maintain telomere length throughout organismal lifespan. Consequently, telomere shortening occurs along with physiological aging in humans and mice and this process is proposed to underlie aging and age-associated diseases as well as organismal longevity25,26.\n\nIn addition to the core components TERT and TERC, the telomerase holoenzyme further consists of the accessory dyskerin complex composed of the proteins DKC1, NOP10, NHP2, and GAR127,28, which also play essential roles in telomere biology. Holoenzyme assembly is thought to occur in the Cajal bodies29, and subsequently TCAB1 and TPP1 are required for proper trafficking of telomerase to telomeres. Moreover, the discovery of a long non-coding telomeric repeat-containing RNA, TERRA30,31, which has been proposed to regulate various aspects of telomere function, adds yet another level of complexity to telomere regulation32,33. Another crucial issue in telomere stability and maintenance is the replication of telomeric DNA, for which a myriad of proteins are required. Key factors in telomeric DNA replication are the CST complex (comprising the proteins CTC1, STN1, and TEN1)34,35, which facilitates telomere replication and simultaneously limits telomerase activity. WRN is a helicase with 3′ to 5′ exonuclease activity, which is also required for efficient telomere replication36 as well as processing of the 3’ telomeric overhang37,38. The helicase BLM contributes to telomere stability by resolving late replication structures39, whereas FEN1 and RTEL1 function in Okazaki fragment processing40 and T-loop disassembly during replication41, respectively. We recently published an in-depth review on the role of these proteins in telomere replication including the consequences for telomere maintenance if their function is impaired42.\n\nIn this review, we will discuss the role of telomeres in the origin of age-associated diseases and organismal longevity, as well as the potential use of telomerase as a therapeutic target to delay aging and to prevent and treat age-related diseases.\n\n\nTelomeres as hallmarks of aging and longevity\n\nAging is a multifactorial process that results in a progressive functional decline at cellular, tissue, and organismal levels. During recent years, a number of molecular pathways have been identified as main molecular causes of aging, including telomere attrition, cellular senescence, genomic instability, stem cell exhaustion, mitochondrial dysfunction, and epigenetic alterations, among others26. Interestingly, telomere attrition is considered a primary cause of aging, as it can trigger all the above-mentioned hallmarks of aging, although the degree to which it is a principal cause of aging is under active investigation26. Critical telomere shortening elicits the induction of cellular senescence or the permanent inability of cells to further divide, which in turn has been proposed to be at the origin of different disease states17,43. In addition, telomere attrition in the stem cell compartments results in the exhaustion of their tissue- and self-renewal capacity, thus also leading to age-related pathologies44,45. Indeed, when this telomere exhaustion occurs prematurely owing to germline mutations in telomere maintenance genes (i.e. telomerase or shelterin genes), this triggers a premature loss of the renewal capacity of tissues leading to the so-called telomeropathies or telomere syndromes, including aplastic anemia and pulmonary fibrosis, among others46–49. Loss of DNA damage checkpoints can also allow the propagation of cells with short/damaged telomeres, thus leading to chromosome end-to-end fusions and genomic instability, as well as age-associated diseases like cancer50,51. A link between dysfunctional telomeres and mitochondrial compromise has been also proposed through transcriptional repression of the PGC-1α and PGC-1β genes by short telomeres, thus linking dysfunctional telomeres to mitochondrial aging52. Finally, short telomeres can trigger epigenetic changes at telomeric as well as subtelomeric chromatin53. In this regard, epigenetic regulation of telomeres has been described in processes that involve de-differentiation and loss of cellular identity such as during tumorigenesis54, as well as during the induction of pluripotency55. In particular, loss of heterochromatic marks at telomeres results in telomere elongation and increased telomere recombination53.\n\nOf note, in addition to the persistent DNA damage response elicited by critically short telomeres, it recently became evident that a large proportion of DNA damage in stress-induced senescence resides in telomeres. Importantly, this DNA damage is independent of telomere length and accumulates with aging in primates and mice, suggesting that stress-induced and telomere length-independent senescence may contribute to the aging process too56,57.\n\nIn addition to being considered a primary molecular cause of aging, telomere shortening with time has been proposed to be a biomarker of biological aging, with a potential prognostic value for many different age-associated diseases, including cardiovascular failure58–64. Interestingly, telomere length has also been proposed as a marker of longevity. A study longitudinally following telomere length throughout the lifespan of individual zebra finches demonstrated that telomere length at day 25 after birth is a strong predictor of individual lifespan in this species65. In mice, a similar longitudinal follow up of telomere length throughout lifespan showed the rate of increase of short telomeres with time but not average telomere length or the rate of telomere shortening was predictive of individual lifespan66. This study also showed for the first time that laboratory wild-type mice shortened telomeres at a pace that was 100-fold faster than humans, thus providing a potential explanation for shorter lifespans in mice (2–3 years) compared to humans, in spite of their long telomere length at birth (~50–150 kb in mice versus ~15–20 kb in humans)67,68. A similar scenario was found in dogs, where telomere shortening has been described to be 10-fold faster than in humans69. These findings suggest that it is the ability of different species to maintain telomeres rather than average telomere length per se that may be determinant of species longevity. This idea is further supported by longitudinal studies in free-living birds. In particular, in Seychelles warblers, telomeres shorten throughout life and higher rates of telomere shortening predict mortality70. Similarly, survival in jackdaws can be predicted by nestling telomere shortening but not by absolute telomere length71.\n\nAdditional and independent evidence that the ability to maintain telomeres may determine mouse longevity came from the description of an age-specific metabolic signature predictive of chronological age in wild-type mice72. In particular, when this signature was used to predict the age of either telomerase-deficient or TERT-overexpressing mice, it predicted older or younger ages than their chronological age, respectively, in agreement with shorter telomeres and shorter lifespan in the telomerase-deficient mice, and longer telomeres and extended lifespan in the TERT-overexpressing mice72, thus suggesting that telomere length is a determinant of aging in wild-type mice.\n\nIn humans, a large number of cross sectional epidemiological studies confirmed telomere shortening with aging in humans16,73. Recently published data from the GERA cohort (Genetic Epidemiology Research on Adult Health and Aging), which comprises more than 100,000 individuals, further confirmed this correlation and also showed that telomere length correlates positively with survival in subjects older than 75, i.e. longer telomeres provide more years of life74. This is in agreement with a previous report showing that telomere length positively correlates with better median survival in individuals who are 60 years of age or older75. However, contradictory reports exist which do not support the correlation between average telomere length and the prediction of remaining years of life in the old and oldest76,77. In this regard, lessons from other species (mice, birds) show the importance of determining not only average telomere length but also longitudinal changes in telomere length as well as changes in the abundance of short telomeres. Thus, future epidemiological studies should take individual telomeres and their change over time into account (i.e. the rate of increase of the fraction of short telomeres). In this regard, methods that can quantify the presence of short telomeres, like the high-throughput quantitative telomere fluorescence in situ hybridization (FISH) technique58 or single telomere length analysis (STELA)78 will be important for establishing telomere shortening as a biomarker of human aging.\n\n\nIntrinsic and environmental instigators of telomere length\n\nAs mentioned above, there are differences in the pace of telomere shortening across species, which indeed may contribute to explaining their different longevities, at least in part. The average telomere shortening in human blood cells occurs at a rate of 31–72 base pairs per year79,80 while mouse telomeres shorten around a hundred times faster than that66. This indicates that, in addition to the intrinsic end replication problem, there are other factors contributing to telomere attrition. In particular, oxidative damage may severely impact on telomere length. Cells exposed to oxidative stress conditions (e.g. H2O2, chronic hyperoxia) display accelerated telomere shortening and reduced replicative lifespans, whereas antioxidant treatment has the opposite effect81. In humans, the choice of lifestyle can influence telomere shortening. As an example, smoking, an unhealthy diet (e.g. high cholesterol, alcohol intake), or obesity might lead to telomere shortening by provoking tissue inflammation and oxidative stress82–87. Moreover, accelerated telomere shortening in leukocytes has been associated with psychological stress. In particular, patients with depression disorders have shorter telomeres compared to healthy individuals88, and this telomere erosion is found in all lymphocyte subpopulations of the adaptive immune system89. Stress provoked by physical abuse of children has been also associated with telomere shortening90. Furthermore, there is a wealth of studies investigating telomere length in major depressive disorder (MDD), a severe illness which shows signs of premature aging60,91,92. In particular, it has been described that telomere length in MDD subjects corresponds to a 10-year increase in biological age93 compared to healthy subjects. In line with this, increased abundance of short telomeres in patients with bipolar II disorder has also been described to correspond to a 13-year older biological age, again in agreement with increased risk for developing different diseases in these patients94. Interestingly, shorter telomeres are also associated with cognitive impairment in the elderly58.\n\nIn contrast to the detrimental factors causing accelerated telomere shortening, certain life habits (e.g. a diet rich in omega-3 fatty acids)81,95, as well as physical activity, exercise, and fitness, have been proposed to reduce telomere erosion and thus slow down the pace of aging96–98.\n\nIn addition to these various intrinsic and environmental factors, telomere length is also dictated by a genetic component. Earlier twin and family studies and a recent meta-analysis comprising nearly 20,000 subjects demonstrate that telomere length is highly heritable79,99–101. Whether the inheritance of telomere length correlates more strongly with paternal or maternal telomere length, however, is still debated102. Interestingly, in another twin study Christensen and colleagues reported that the perceived age in twins older than 70 years of age is a robust biomarker of aging which strongly correlates with telomere length. Moreover, within twin pairs, the twin with greater telomere length tends to look younger and live longer103.\n\n\nGenetic models to understand the causal role of telomeres in disease and longevity\n\nFirm experimental demonstration that critical telomere shortening is causative of aging was first achieved by generating mice deficient for telomerase. Mice deficient for TERC have progressively shorter telomeres over generations, leading to chromosome instability, developmental defects, premature aging phenotypes, and ultimately mouse infertility and premature death104–106. These mice show a decreased median and maximum lifespan already at the first generation107, and this decreased longevity and associated aging pathologies are anticipated with each mouse generation, thus demonstrating that telomere length in mice is causal of aging and longevity. Importantly, restoration of TERC expression in mice with inherited critically short telomeres is sufficient to prevent the phenotypes associated with short telomeres in these mice, including aplastic anemia, intestinal atrophy, and infertility, among others108,109. In agreement with these pioneer studies, genetic ablation of TERT was shown to have similar consequences on organismal aging and lifespan110,111. Furthermore, TERT reconstitution in late generation TERT-deficient mice also led to telomere elongation, lower DNA damage load, and reversal of degenerative phenotypes in these mice112. In line with these findings, lack of telomerase in lower vertebrates such as the zebrafish also causes premature aging which can be rescued by either telomerase restoration or inhibition of p53, which signals telomere damage113. Together, these findings demonstrate that short telomeres are causative of aging and that premature aging specifically induced by telomerase deficiency and short telomeres can be rescued by telomerase re-expression.\n\nIn line with mouse studies, a number of human syndromes were later described to be caused by germ line mutations in telomerase and shelterin genes, the so-called telomere syndromes47. As in the telomerase-deficient mouse model, the diseases associated with telomerase mutations are anticipated with increasing generations and involve a loss of the ability of tissues to regenerate, resulting in skin abnormalities, aplastic anemia, or pulmonary fibrosis46,47. These analogies between humans and mice highlight that telomere length as a genetic determinant of disease and longevity is a molecular mechanism conserved in these species.\n\nHowever, definitive genetic demonstration that telomere length is also causative of physiological aging in normal individuals first came from telomerase overexpression studies in mice. In particular, mice with increased transgenic telomerase expression throughout their lifespans were able to maintain longer telomeres with aging, showed decreased molecular (i.e. lower DNA damage) and physiological biomarkers of aging, showed a delayed appearance of age-related pathologies (osteoporosis, metabolic decline, etc.), and showed a significant increase in organismal longevity. In particular, transgenic TERT overexpression in mice engineered to be cancer resistant resulted in decreased incidence of aging-related pathologies and a striking 40% extension of median survival compared to wild-type mice114. This study demonstrated for the first time in any organism the anti-aging activity of telomerase. Importantly, these findings led to the idea that potential therapeutic strategies based on transiently increased telomerase expression could also delay age-associated pathologies and increase longevity. This was first achieved by delivering TERT using non-integrative gene therapy vectors (adeno-associated vectors [AAVs]) into middle-aged and old mice, which resulted in transiently increased TERT expression in the majority of mouse tissues. Importantly, a single treatment with these vectors resulted in elongated telomeres in a range of organs, delayed age-associated pathologies, and significantly extended median and maximal lifespan in both age groups115. Moreover, these mice did not show increased cancer; instead, as seen in other age-related conditions, cancer was also delayed115. Thus telomere-based gene therapies using non-integrative vectors may represent a new therapeutic strategy to transiently activate TERT for the prevention or treatment of many different age-related pathologies (see below).\n\n\nTelomeres and Telomerase as therapeutic targets\n\nA substantial number of companies are now aiming to harness the knowledge that has been generated, unveiling the molecular mechanisms of aging in order to develop a new class of drugs to prevent and treat the major age-related diseases116. In this regard, telomerase overexpression studies in mice have been proof of principle that just modifying a single hallmark of aging, i.e. telomere shortening, this was sufficient to delay not one but many different age-associated pathologies in mice, including cognitive decline114,115. Indeed, the use of telomerase activation in delaying aging-associated conditions has spurred the interest of commercial enterprises. For instance, the low-potency telomerase activator TA-65 (a bio-active compound isolated from the herb Astragalus membranaceus) has been shown to lead to a mild increase in telomere length in mice117, zebra finches118, and humans119, and to improve several aging-related parameters in mice and humans117,119, although no increase in longevity has been reported in longitudinal mouse studies117. On the other hand, other natural compounds like sex hormones have been found to activate TERT at the transcriptional level120–122. In this regard, androgen therapy has been applied as a first-line treatment in aplastic anemia for decades with mixed success and without a clear understanding of the mechanism that underlies remission in some patients but not in others123,124. A recent study in mice which develop full-blown aplastic anemia provoked by short telomeres showed that androgen therapy rescues telomere attrition and subsequent death from aplastic anemia122, indicating that telomerase activation may indeed be a treatment option for diseases associated with flawed telomere maintenance (i.e. telomeropathies or telomere syndromes). However, potential off-target effects of compounds that activate TERT at a transcriptional level should be a concern. In particular, TA-65 has been shown to activate TERT through activation of mitogenic pathways that lead to the activation of the oncogene c-myc117,125 and thus may drive cancer. Interestingly, such off-target effects may be circumvented through direct delivery of TERT, such as by means of systemic gene therapy using non-integrative AAV vectors, which showed a significant delay of age-related pathologies in mice and increased longevity115. A recent study using fibroblasts in vitro also proposed delivery of the TERT mRNA as a way to activate telomerase126. However, it should be mentioned that strategies for telomerase activation, indirect or direct, have raised safety concerns due to the close correlation of most cancers and constitutive reactivation of endogenous telomerase. This highlights that, in addition to proof-of-concept studies in mice, the development of safe strategies for transient and controllable telomerase activation in humans should be a future goal.\n\nIn this regard, TERT gene therapy with AAVs is particularly attractive for TERT activation, since the non-integrative and replication-incompetent properties of AAVs allow for cell division-associated telomere elongation and subsequent loss of TERT expression as cells divide, thus restricting TERT expression to a few cell divisions. Thus, this strategy assures a transient and relatively genome-safe TERT activation. In contrast, the use of TERT mRNA currently lacks appropriate systems for in vivo delivery, and thus its use may be restricted to ex vivo applications.\n\nIt is likely that the first clinical use of a TERT-based therapy, such as the TERT gene therapy approach developed by us, will be for the treatment of the human telomere syndromes, including aplastic anemia and pulmonary fibrosis. However, this requires the development of appropriate preclinical models and the subsequent clinical trials in humans. In this regard, we have recently generated two mouse models which recapitulate the clinical features of aplastic anemia127 and pulmonary fibrosis128. The disease in both models is provoked by short and dysfunctional telomeres and thus these models provide a platform for further testing of TERT-based treatment strategies for the telomere syndromes.\n\nGiven that physiological aging is provoked, at least in part, by telomere shortening, a TERT gene therapy may be used not only for the prevention and treatment of telomere syndromes but also for the treatment of multiple age-related diseases. In this regard, short telomeres have been extensively associated with a higher risk for cardiovascular disease64,129,130. In support of a potential use of TERT activation in the treatment of age-related diseases, we demonstrated that TERT gene therapy can efficiently rescue mouse survival and heart scarring in a preclinical mouse model for heart failure upon induction of acute myocardial infarction131.\n\nCollectively, experiments in cell and animal models provide proof of concept for the feasibility of telomerase activation approaches to counteract telomere shortening and its consequences (Figure 1). In particular, the successful use of telomerase gene therapy in animal models of aging and short telomere-related diseases paves the way for the development of therapeutic telomerase treatments in human aging and associated disease.\n\nTelomere shortening is a life-long process that is influenced by a number of intrinsic and environmental factors that either accelerate or slow down natural telomere attrition, which causes aging and the emergence of age-related diseases. The identification of telomere shortening as a driver of molecular aging has triggered the development of telomerase-based strategies to (re)elongate telomeres and thus to delay aging and associated disease. Abbreviations: AAV, adeno-associated vectors; TERT, telomerase reverse transcriptase.", "appendix": "Competing interests\n\n\n\nMaria A. Blasco is co-founder of Life Length, a biotechnology company that commercializes measurement of telomere length for different applications.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nde Lange T: Protection of mammalian telomeres. Oncogene. 2002; 21(4): 532–40. PubMed Abstract \| Publisher Full Text\n\nde Lange T: Shelterin: the protein complex that shapes and safeguards human telomeres. Genes Dev. 2005; 19(18): 2100–10. 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PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nBroer L, Codd V, Nyholt DR, et al.: Meta-analysis of telomere length in 19,713 subjects reveals high heritability, stronger maternal inheritance and a paternal age effect. Eur J Hum Genet. 2013; 21(10): 1163–8. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nEisenberg DT: Inconsistent inheritance of telomere length (TL): is offspring TL more strongly correlated with maternal or paternal TL? Eur J Hum Genet. 2014; 22(1): 8–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChristensen K, Thinggaard M, McGue M, et al.: Perceived age as clinically useful biomarker of ageing: cohort study. BMJ. 2009; 339: b5262. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nBlasco MA, Lee HW, Hande MP, et al.: Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. Cell. 1997; 91(1): 25–34. PubMed Abstract \| Publisher Full Text\n\nHerrera E, Samper E, Blasco MA: Telomere shortening in mTR-/- embryos is associated with failure to close the neural tube. EMBO J. 1999; 18(5): 1172–81. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee HW, Blasco MA, Gottlieb GJ, et al.: Essential role of mouse telomerase in highly proliferative organs. Nature. 1998; 392(6676): 569–74. PubMed Abstract \| Publisher Full Text\n\nGarcía-Cao I, García-Cao M, Tomás-Loba A, et al.: Increased p53 activity does not accelerate telomere-driven ageing. EMBO Rep. 2006; 7(5): 546–52. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSamper E, Flores JM, Blasco MA: Restoration of telomerase activity rescues chromosomal instability and premature aging in Terc-/- mice with short telomeres. EMBO Rep. 2001; 2(9): 800–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHemann MT, Strong MA, Hao LY, et al.: The shortest telomere, not average telomere length, is critical for cell viability and chromosome stability. Cell. 2001; 107(1): 67–77. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nLiu Y, Snow BE, Hande MP, et al.: The telomerase reverse transcriptase is limiting and necessary for telomerase function in vivo. Curr Biol. 2000; 10(22): 1459–62. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nErdmann N, Liu Y, Harrington L: Distinct dosage requirements for the maintenance of long and short telomeres in mTert heterozygous mice. Proc Natl Acad Sci U S A. 2004; 101(16): 6080–5. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nJaskelioff M, Muller FL, Paik JH, et al.: Telomerase reactivation reverses tissue degeneration in aged telomerase-deficient mice. Nature. 2011; 469(7328): 102–6. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nAnchelin M, Alcaraz-Pérez F, Martínez CM, et al.: Premature aging in telomerase-deficient zebrafish. Dis Model Mech. 2013; 6(5): 1101–12. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nTomás-Loba A, Flores I, Fernández-Marcos PJ, et al.: Telomerase reverse transcriptase delays aging in cancer-resistant mice. Cell. 2008; 135(4): 609–22. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nBernardes de Jesus B, Vera E, Schneeberger K, et al.: Telomerase gene therapy in adult and old mice delays aging and increases longevity without increasing cancer. EMBO Mol Med. 2012; 4(8): 691–704. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nScott CT, DeFrancesco L: Selling long life. Nat Biotechnol. 2015; 33(1): 31–40. PubMed Abstract \| Publisher Full Text\n\nBernardes de Jesus B, Schneeberger K, Vera E, et al.: The telomerase activator TA-65 elongates short telomeres and increases health span of adult/old mice without increasing cancer incidence. Aging Cell. 2011; 10(4): 604–21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nReichert S, Bize P, Arrivé M, et al.: Experimental increase in telomere length leads to faster feather regeneration. Exp Gerontol. 2014; 52: 36–8. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nHarley CB, Liu W, Blasco M, et al.: A natural product telomerase activator as part of a health maintenance program. Rejuvenation Res. 2011; 14(1): 45–56. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKyo S, Takakura M, Kanaya T, et al.: Estrogen activates telomerase. Cancer Res. 1999; 59(23): 5917–21. PubMed Abstract\n\nCalado RT, Yewdell WT, Wilkerson KL, et al.: Sex hormones, acting on the TERT gene, increase telomerase activity in human primary hematopoietic cells. Blood. 2009; 114(11): 2236–43. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBär C, Huber N, Beier F, et al.: Therapeutic effect of androgen therapy in a mouse model of aplastic anemia produced by short telomeres. Haematologica. 2015; 100(10): 1267–74. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShahidi NT, Diamond LK: Testosterone-induced remission in aplastic anemia of both acquired and congenital types. Further observations in 24 cases. N Engl J Med. 1961; 264: 953–67. PubMed Abstract \| Publisher Full Text\n\nJaime-Pérez JC, Colunga-Pedraza PR, Gómez-Ramírez CD, et al.: Danazol as first-line therapy for aplastic anemia. Ann Hematol. 2011; 90(5): 523–7. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nMolgora B, Bateman R, Sweeney G, et al.: Functional assessment of pharmacological telomerase activators in human T cells. Cells. 2013; 2(1): 57–66. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRamunas J, Yakubov E, Brady JJ, et al.: Transient delivery of modified mRNA encoding TERT rapidly extends telomeres in human cells. FASEB J. 2015; 29(5): 1930–9. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nBeier F, Foronda M, Martinez P, et al.: Conditional TRF1 knockout in the hematopoietic compartment leads to bone marrow failure and recapitulates clinical features of dyskeratosis congenita. Blood. 2012; 120(15): 2990–3000. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPovedano JM, Martinez P, Flores JM, et al.: Mice with Pulmonary Fibrosis Driven by Telomere Dysfunction. Cell Rep. 2015; 12(2): 286–99. PubMed Abstract \| Publisher Full Text \| Faculty Opinions Recommendation\n\nFyhrquist F, Saijonmaa O, Strandberg T: The roles of senescence and telomere shortening in cardiovascular disease. Nat Rev Cardiol. 2013; 10(5): 274–83. PubMed Abstract \| Publisher Full Text\n\nHaycock PC, Heydon EE, Kaptoge S, et al.: Leucocyte telomere length and risk of cardiovascular disease: systematic review and meta-analysis. BMJ. 2014; 349: g4227. PubMed Abstract \| Publisher Full Text \| Free Full Text \| Faculty Opinions Recommendation\n\nBär C, Bernardes de Jesus B, Serrano R, et al.: Telomerase expression confers cardioprotection in the adult mouse heart after acute myocardial infarction. Nat Commun. 2014; 5: 5863. PubMed Abstract \| Publisher Full Text" }	[ { "id": "12012", "date": "21 Jan 2016", "name": "Jerry W Shay", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12013", "date": "21 Jan 2016", "name": "Fabrizio d'Adda di Fagagna", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12014", "date": "21 Jan 2016", "name": "Lucio Comai", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12015", "date": "21 Jan 2016", "name": "F. Bradley Johnson", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nUntil it is demonstrated that enhanced telomere maintenance delays or reverses age-related pathologies in humans, it will not be know for certain to what extent telomere dysfunction contributes to natural human aging. Nonetheless, the current evidence, as reviewed by Bar and Blasco, does suggest that targeting telomeres will prove to be helpful for the amelioration of age-related diseases.", "responses": [] } ]	1	https://f1000research.com/articles/5-89
https://f1000research.com/articles/5-88/v1	20 Jan 16	{ "type": "Review", "title": "Can follicular helper T cells be targeted to improve vaccine efficacy?", "authors": [ "Michelle A. Linterman", "Danika L. Hill", "Danika L. Hill" ], "abstract": "The success of most vaccines relies on the generation of antibodies to provide protection against subsequent infection; this in turn depends on a robust germinal centre (GC) response that culminates in the production of long-lived antibody-secreting plasma cells. The size and quality of the GC response are directed by a specialised subset of CD4+ T cells: T follicular helper (Tfh) cells. Tfh cells provide growth and differentiation signals to GC B cells and mediate positive selection of high-affinity B cell clones in the GC, thereby determining which B cells exit the GC as plasma cells and memory B cells. Because of their central role in the production of long-lasting humoral immunity, Tfh cells represent an interesting target for rational vaccine design.", "keywords": [ "T follicular helper cells", "vaccines", "immunity", "antibody" ], "content": "Introduction\n\nVaccination is one of the most successful, cost-effective interventions for combating infectious disease, thereby reducing infection-related disease, disability and death worldwide1. Despite this enormous success, there are still multiple infections that require a vaccination solution, including vaccines that protect against HIV and malaria2,3, and a way to improve vaccine efficacy in older persons4. The majority of current vaccines have been developed empirically rather than rationally, suggesting that a change in approach to vaccine development may enable breakthroughs in vaccine design5. All routine human vaccinations, with the exception of the Bacillus Calmette-Guérin tuberculosis vaccine, provide protection by generating antibodies that block the ability of a pathogen to establish an infection and that target it for destruction. Vaccine-induced antibody responses are supported by T follicular helper (Tfh) cells; here, we discuss how advances in the knowledge of Tfh cell biology could be used to improve vaccine efficacy.\n\nThe production of vaccine-specific antibodies can occur via two cellular routes: the extrafollicular or germinal centre (GC) responses. The extrafollicular response produces an initial burst of antibodies early after immunisation and can occur with or without T cell help6. These extrafollicular plasma cells are short-lived and because of this are not able to provide a long-term source of protective antibodies7. The GC is a specialised microenvironment that forms in secondary lymphoid tissues after immunisation when antigen-activated B cells migrate to the B cell follicle, begin to proliferate, and undergo somatic hypermutation (SHM) of their immunoglobulin genes8. The cellular products of the GC are long-lived plasma and memory B cells that can provide protection for decades after initial exposure9. Because of the longevity of these cells, the GC represents an exciting target to improve vaccine responses in situations in which there is an unmet clinical need.\n\nThe GC is a microenvironment of intense cellular collaboration: GC B cells, Tfh cells, T follicular regulatory (Tfr) cells, tingible body macrophages and follicular dendritic cells (FDCs) act together to generate a robust response (Figure 1). The “multi-player” nature of the GC means that there are a number of cellular targets that can be manipulated in the GC during vaccination in an attempt to modulate its output. Here, we will discuss whether targeting Tfh and Tfr cells may be a successful strategy for improving the GC in response to vaccination.\n\nThe germinal centre (GC) is initiated when activated B cells migrate into the B cell follicle, begin to divide, and mutate their B-cell receptor (BCR) genes. These proliferating centroblasts then exit the cell cycle, becoming centrocytes that use their mutated BCR to collect antigen from the surface of follicular dendritic cells (FDCs) and present processed peptide on major histocompatibility complex class II to T follicular helper (Tfh) cells. Tfh and T follicular regulatory (Tfr) cell differentiation initiates during T cell priming when Bcl-6, the transcription factor required for Tfh and Tfr cell differentiation, is upregulated. Subsequent interactions with B cells stabilise Bcl-6 expression in pre-Tfh and pre-Tfr cells, allowing them to migrate into the GC where Tfh cells provide help to centrocytes and Tfr cells act as GC suppressors. As an alternative to entering the GC, antigen-activated B cells can also receive T cell help and differentiate into short-lived extrafollicular plasma cells that produce the first wave of protective antibodies. DC, dendritic cell; FDC, follicular dendritic cell; mB, memory B cell; SHM, somatic hypermutation; TBM, tingible body macrophage; Th, T helper cell; Treg, regulatory T cell.\n\n\nFollicular T cells and the GC response\n\nIn physiological conditions, the GC is absolutely dependent on T cell help for its formation. Experimentally, immunisation with superantigens can initiate GC formation in the absence of T cells, however, the response collapses after five days, demonstrating the absolute requirement of T cells for the GC to produce plasma cells10. The specific requirement for T cell help is fulfilled by a specialised subset of CD4+ helper T cells: Tfh cells. Tfh cells differentiate in response to immunisation by using a specific differentiation pathway that requires various cytokine signals and multiple rounds of antigen presentation that culminates in the expression of the transcriptional repressor Bcl-6 and localisation to the GC11. The signals that are required for Tfh cell development have been well characterised in both humans and mice; these are summarised in Table 112–42. These pathways may represent viable targets to manipulate the number of Tfh cells that form after vaccination.\n\nFor each receptor expressed by T follicular helper (Tfh) cells or their precursors, the predominant downstream signalling pathway is indicated. Surface receptors and signals were deemed to influence Tfh cell development if Tfh cell frequencies were altered in deficient mice or in human patients with genetic deficiency. ↑, an increase in T follicular helper frequencies; ↓, a decrease in T follicular helper frequencies; =, no change in T follicular helper frequencies; DC, dendritic cell; ND, not determined.\n\nAfter establishment of the GC, it divides into two distinct zones: the GC B cells rapidly divide in the dark zone and undergo SHM of their B-cell receptor (BCR) genes and then exit the cell cycle and migrate to the light zone of the GC, where the fully differentiated Tfh cells are located. The random nature of SHM requires that mutated B cells undergo a selection process before they exit the GC as plasma cells or memory B cells. The GC B cells test their newly mutated BCR by collecting antigen-containing immune complexes from the surface of FDCs and then present processed antigen to Tfh cells on major histocompatibility complex (MHC) class II43. The B cells that are able to present the most antigen to Tfh cells are the recipients of T cell help44, which results in the upregulation of c-myc and subsequent return to the dark zone to undergo further rounds of proliferation and mutation45–47. By this mechanism, Tfh cells act as a limiting factor in the selection of high-affinity GC B cells. This could simply be numerical, as B cells outnumber Tfh cells in the GC. Alternatively, there may be an interaction threshold that needs to be overcome before Tfh and B cells form productive conjugates in the GC. During Tfh and B cell interactions in the GC, there is a bidirectional exchange of signals: Tfh cells provide help in the form of CD40L, interleukin-21 (IL-21) and IL-4 to GC B cells, which supports proliferation and survival, while B cells provide inducible T cell co-stimulator ligand (ICOSL) to Tfh cells48,49. Thus, Tfh cells facilitate the preferential expansion and mutation of high-affinity GC B cell clones and are key regulators of the size and quality of the GC response.\n\nIn addition to Tfh cells, there is a second specialised subset of CD4+ T cells present in the GC: Tfr cells. Tfr cells derive from Foxp3+ regulatory T (Treg) cells and act as suppressors of the GC response. Tfr cells limit the size of the GC response, thereby acting as a counterbalance to Tfh cells50–52. Although there are some transcriptional and phenotypic similarities between Tfh and Tfr cells, functionally Tfr cells are suppressive and thus resemble conventional Treg cells53.\n\nTogether, Tfh and Tfr cells are key regulators of the GC response; Tfh cells positively control the size and output of GC, whereas Tfr cells act as negative regulators of the response. This suggests that strategies to enhance Tfh number or function (or both) or reduce Tfr cells may enhance GC responses and promote a more potent response to vaccination.\n\n\nCirculating peripheral blood Tfh as biomarkers of GC Tfh cells\n\nThe majority of advances in Tfh and Tfr cell biology have occurred through studying secondary lymphoid tissues in mice. This is largely because of the impressive range of tools (e.g., genetically modified mice) that allow precise dissection of GC biology in mice and because access to healthy human lymphoid tissue can be difficult, particularly for the purpose of studying GC responses to a defined antigenic stimulus. In an attempt to circumvent this issue, a circulating cellular biomarker of GC Tfh cells has been used to further investigate Tfh cell biology in humans. These cells have been coined circulating Tfh-like (cTfh) cells and were first identified in sanroque mice and in patients with systemic lupus erythematous and were defined by CXCR5, programmed cell death protein 1 (PD-1), and ICOS expression54. Subsequently, it has been shown that an increased frequency of cTfh cells coincides with the peak GC response in mice and the plasmablast response to influenza vaccination in humans55,56. These studies suggest that cTfh cells may be a key tool for studying the role of Tfh cells in human vaccine responses. However, the use of cTfh cells as a surrogate of GC Tfh cell responses in humans requires a robust assessment of the strengths and limitations of this approach.\n\nStudies in both humans and mice support a link between the GC Tfh and cTfh cells. Human cTfh cells can provide help to B cells in vitro and upon stimulation display several features consistent with GC Tfh cells, including ICOS expression and expression of IL-21 and CXCL1357–59. Although cTfh cells do not express BCL6, they have low levels of BLIMP1 and express cMAF, and this indicates that they share features of transcriptional control with GC Tfh cells57–59. Several human immunodeficiency syndromes that are associated with severely impaired GC responses due to loss of functional CD40L60, ICOS15,61, STAT362 or IL-12βR136 display corresponding reductions in blood cTfh cells, suggesting that cTfh cells can be a biomarker for an active GC response. Conversely, mice deficient for Sh2d1a have impaired GC reactions but unchanged cTfh frequencies55. Consistent with this, patients with X-linked lymphoproliferative disease (XLP) caused by defects in SH2D1A, or healthy XLP carriers, did not display alterations in cTfh frequencies55. These data suggest that although cTfh cells resemble GC Tfh cells, a GC reaction is not required for cTfh cell development, which parallels the development of extrafollicular Tfh cells63. One possibility is that cTfh cells are memory cells that are induced upon vaccination to enable fast GC Tfh responses following subsequent infection. Consistent with this idea, tetanus- and smallpox-specific cTfh cells can be identified in humans years after vaccination58,64. In mice, cTfh cells have the capacity to become GC Tfh cells and support the GC response55,65, suggesting that cTfh cells may be an important component of secondary immune responses and therefore a biologically relevant cell population in successful vaccination. Despite the recent surge in correlative studies assessing cTfh cells in a multitude of disease settings, unsupervised comparisons of gene expression in GC Tfh cells have not been performed in blood and lymphoid tissue samples from the same individual58, and antigen-specific responses have not been determined. Addressing these issues will help to clarify the relationship between circulating and GC Tfh cells.\n\nAn interesting feature of GC Tfh cells is their well-described heterogeneity66, and cTfh cells are not an exception. Analysis of blood CD4+CXCR5+ cells for expression of PD1, CCR7, CXCR3, CCR6 and ICOS has been proposed to define nine populations of cTfh cells67. However, across the range of studies, robust B cell helper function in vitro has consistently been demonstrated for CD4+CXCR5+ cells that express high levels of PD-1 or ICOS or both67. CXCR3 and CCR6 expression on cTfh enables identification of cTfh cells with Th1-like (cTfh1, CXCR3+CCR6−), Th2-like (cTfh2, CXCR3−CCR6−) and Th17-like (cTfh17, CXCR3−CCR6+) properties, including the expression of transcription factors and cytokines that define these T helper subsets57. cTfh2 and cTfh17 can support naïve and memory B cells to produce antibodies in vitro, whereas cTfh1 cells have limited in vitro helper function57,58, although following influenza vaccination a population of ICOS+ cTfh1 cells were able to help memory B cells make antibodies56. One limitation of these studies is that it remains unclear to what extent in vitro B cell helper function reflects effective GC Tfh help in vivo. Although these cTfh cell subtypes have been identified in blood, characterisation of GC Tfh cell populations by using these markers has been limited, calling into question the relevance of these subsets to GC biology. However, tonsillar Tfh can co-express BCL6 and RORγt67 and a proportion of human lymph node Tfh cells express CXCR3 (D.L. Hill, unpublished), and this suggests that comparable heterogeneity exists within in the GC Tfh cell population. But whether there is a specialised role for Th1/Th2/Th17 polarised GC Tfh cells in the GC has yet to be elucidated.\n\nThe polarisation of GC Tfh cells depends on the stimuli provided during differentiation. In mice, Th2-biased infections produce IL-4-secreting GC Tfh cells, whereas Th1-biased infections support interferon-gamma-positive (IFNγ+) GC Tfh cells68–71. In humans, cTfh2 cell frequency increases in people with Th2-polarised Schistosoma japonicum infection72, whereas cTfh1 cells are preferentially expanded during Th1-biased acute Plasmodium falciparum infection and after seasonal influenza vaccination56,73. Thus, different cytokine environments induced by specific infections or immunisations appear to drive Tfh cell polarisation and may enable Tfh cells to appropriately support B cell production of the antibody isotype required to clear the infection. For example, in mice, IFNγ+ Tfh cells could be found in conjugates with Ig2a+ B cells, whereas IL-4+ Tfh cells were more likely to be paired with IgG1+ B cells74. Immunity against pathogens relies upon production of specific antibody isotypes that ultimately play an important role in clearing infections. For example, inappropriate production of Th1-supported isotypes to the parasitic roundworm Wuchereria bancrofti75 and Th2-supported isotypes in P. falciparum malaria76 correlates with poor disease outcomes. Therefore, cTfh cell heterogeneity may reflect the ability of Tfh cells to be shaped by the environmental signals present during differentiation, which enables them to guide an appropriate B cell response to infection or vaccination, to facilitate pathogen clearance.\n\nIt has been proposed that the limited efficacy of seasonal influenza vaccination results from the preferential induction of cTfh1 cells58. As such, skewing Tfh cells away from Tfh1-like and toward Tfh2/17-like may represent a potential target to enhance antibody titres following influenza vaccination (Figure 2A). Interestingly, blocking the Th1 cytokines IL-2 and tumour necrosis factor (TNF) improved Tfh-mediated B cell help in vitro77,78. However, this approach may not be effective for generating protective responses to vaccination in vivo. Passive transfer of broadly neutralising antibodies to hemagglutinin can protect mice from succumbing to experimental influenza infection. Importantly, for some clones, this protection is conferred only by a Th1 polarised IgG2a antibody and not Th2 polarised IgG1, despite having the same ability to bind hemagglutinin79. This suggests that production of Th1-supported isotypes and the selective induction of Th1-like Tfh cells are likely important for generating protective influenza vaccine responses. Yet as current influenza vaccine formulations fail to generate a protective immune response in up to 30% of vaccine recipients80, further enhancing Tfh cell responses may improve vaccine efficacy.\n\n(A) Altering the balance of different subsets of T follicular helper (Tfh) cells toward Tfh2 and Tfh17 cells may enhance the B cell helper capacity of the Tfh cells within the GC and the production of the necessary antibody isotypes. (B) Increasing the number of Tfh cells may help increase the size and output of the GC response. (C) Reducing the frequency of suppressive T follicular regulatory (Tfr) cells may enhance the output of the GC response. FDC, follicular dendritic cell; IFNγ, interferon-gamma; IL-17A, interleukin-17A; mB, memory B cell.\n\nThe potential use of cTfh cell subsets as a biomarker of vaccine efficacy is an attractive possibility that would be easily amenable to clinical trials. Although further study is needed, mounting evidence supports cTfh cells as a relevant population for the study of vaccine responses in humans. However, it is clear that there are limitations for using cTfh cells to study the GC Tfh cell response, as although they appear to be a biomarker for the GC response, they differ from bona fide GC Tfh cells in a number of aspects, including BCL6 expression and dependence on SAP for their differentiation. Because of this, analysing cTfh cells will likely not become a substitute for studies aimed at understanding GC Tfh cell biology, but rather represents an additional tool to interrogate the human response to vaccination. Research that combines assessment of cTfh cells after vaccination, GC Tfh cells from human secondary lymphoid tissues and mouse vaccination models will enable thorough interrogation of strategies that target Tfh cells to improve vaccine efficacy.\n\n\nPotential avenues for modifying follicular T cells to enhance vaccination efficacy\n\nWithin the GC, Tfh cells support the GC response and Tfr cells negatively regulate the magnitude of the GC. As such, manipulating the frequency of Tfh cells or enhancing their function may improve the GC response. Conversely, reducing the number of Tfr cells or their functional capacity may also increase antibody responses to vaccination. Here, we explore the potential strategies for manipulating these T cell subsets with the view to increase GC output following vaccination.\n\nThe numbers of Tfh cells and GC B cells positively correlate81,82, as do the numbers of cTfh and blood plasmablasts after influenza vaccination in humans56. This suggests that strategies to augment Tfh cell number may be a rational approach to enhance vaccine responses (Figure 2B).\n\nAntigen presentation and recognition are central to Tfh cell differentiation, and hence providing ample antigen may enhance Tfh-driven vaccine responses. Increasing the dose of protein antigen enhances the magnitude of Tfh cell responses in mice82, and in older people a higher dose of seasonal influenza vaccine increases neutralising antibody titres83,84. This increased antigen availability may have enabled increased peptide-MHC II presentation to T cells, resulting in enhanced Tfh cell differentiation and function. Specifically targeting antigen to the CD8+ DC receptor Clec9A enhanced MHC II presentation, antibody responses, Tfh cell numbers and memory B cells in the absence of adjuvants in mice and non-human primates85–87. Likewise, when B cells present high levels of peptide-MHC II to Tfh cells, they are able to gain entry to the GC and, once within the GC, are more likely to be maintained88,89. Consistent with this, the ongoing presence of antigen in people with chronic HIV and hepatitis B virus is associated with expansions in cTfh cells90,91. This suggests that increasing antigen availability may be a strategy to enhance Tfh cell numbers in response to vaccination. However, despite elevated cTfh cells, the majority of chronically HIV-infected individuals fail to mount broadly neutralising antibody responses92. Furthermore, in mice, increasing Tfh cell number by provision of soluble peptide did not increase the frequency of high-affinity B cells in the early phase of the vaccine response93. This suggests that, in some cases, solely increasing Tfh numbers may not be sufficient to enhance vaccine efficacy, and approaches may need to be tailored to the specific vaccination challenge.\n\nOne of the potential challenges to specifically targeting Tfh cells during vaccination is to not perturb normal immune cell homeostasis. The best way to do this has long been considered “the immunologist’s dirty little secret”—adjuvants, which trigger T and B cells to respond to antigen94. Currently, only a handful of different adjuvants have been used in licensed vaccines95, and the use of Alum is the most widespread. Novel or modified adjuvants may prove to be an effective strategy to skew helper T cells to differentiate toward the Tfh cell subset and promote GC responses. The squalene adjuvant MF59 has been shown to increase the quantity, diversity and affinity of antibodies produced following pandemic influenza vaccination96–98. MF59 increases GC B cells, Tfh cells, and antigen-specific DCs following immunisation in mice99,100, and this suggests that MF59 may act via DCs to enhance the GC response. The use of Toll-like receptor (TLR) agonists as adjuvants has been successful in enhancing vaccine responses in mice and non-human primates. Nanoparticles containing TLR4 and TLR7/8 agonists prolonged GC reactions, improved antibody quality, supported memory B cell development in mice101,102, and protected against secondary influenza infection101. It would be particularly pertinent to consider adjuvants that trigger pathways known to enhance Tfh cell differentiation (Table 1). For example, immunisations supplemented with TLR9 agonists enhanced antibody responses and Tfh and GC B cell numbers in mice via DC production of the Tfh-promoting cytokine, IL-633,103. Alternatively, ICOSL binding is a requisite event in multiple stages of Tfh cell development but is not an essential requirement for Th1 or Th2 cell differentiation13,104,105. Upregulation of ICOSL on DCs upon the addition of a TLR2 agonist correlated with enhanced antibody production following protein immunisation in vivo106. Thus, TLR signalling in DCs and B cells could be specifically directed to enhance vaccine antibody and Tfh cell responses.\n\nAnother potential strategy to enhance Tfh cell numbers is to use adjuvants to modulate the cytokines produced by antigen-presenting cells to promote Tfh cell differentiation. In mice, IL-6 and IL-21 support Tfh cell differentiation, whereas IL-2 suppresses Tfh cell fate31,32,35,40,107. In humans, an entirely separate cohort of cytokines support Tfh cells: IL-12, IL-23 and transforming growth factor-beta (TGF-β) (Table 1)36–38. Because of this, the IL-12/STAT4 axis may be a potential target to enhance Tfh cells in humans. However, preclinical trials of an IL-12 expression plasmid adjuvant did not enhance vaccine antibody responses108,109. Alternatively, the addition of Fc-fused IL-7 enhanced B cell and Tfh responses to influenza vaccination in mice and cynomolgus monkeys110, suggesting that delivery of a generic T cell survival signal could be sufficient to enhance the vaccination responses. The different cytokine requirements between mice and humans for Tfh cell differentiation demonstrate the importance of studying human Tfh cell biology.\n\nThe pathways involved in Tfh development are well established (Table 1); however, the signals that regulate Tfh cell effector function are less well described. Increased antigen presentation from B cells increases Tfh cell production of the cytokines IL-4 and IL-2148. These observations suggest that increasing vaccine antigen dose or targeting antigen to B cells may improve Tfh function and the quality of the GC reaction. ICOSL expression by GC B cells promoted calcium-dependent CD40L expression from Tfh cells, and this feed-forward signalling loop provided a competitive advantage to ICOSL-expressing B cells49. Because help via CD40L, IL-21 and IL-4 is important for GC B cell-positive selection, it raises the possibility that adjuvant approaches that lead to increased ICOSL or CD40L in GC B and Tfh cells, respectively, could be a strategy to enhance vaccine responses.\n\nA reduction in the frequency of Tfr cells may be a useful approach to enhance the GC response, particularly in situations such as ageing in which an increased number of Tfr cells correlates with a smaller GC response111. In mice, it is possible to alter the ratio of Tfh to Tfr cells simply by using different adjuvants; the more the ratio favours Tfh cells, the larger the GC response (Figure 2C)103,112. To specifically manipulate Tfr cells, two key inhibitory molecules may be potential targets: cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and PD-1. CTLA-4 is a suppressive mechanism by which Treg and Tfr cells can control GC response to vaccination through limiting CD28 signalling that is important for Tfh maintenance17,113,114. Inhibiting this receptor in mice increases the number of antigen-specific Tfh cells, plasma and memory B cells following immunisation. Proof-of-principle testing could be performed in melanoma patients receiving the CTLA-4 inhibitor ipilimumab and vaccination, although the side effects and cost associated with this compound would prohibit its use with routine vaccines. Another interesting target is PD-1, as signalling through this receptor limits Tfr cell differentiation in mice30. However, this is likely not to be practicable as PD-1 is also expressed highly on Tfh cells115 and targeting PD-1 on Tfr cells specifically would be difficult. Also, some adjuvants support the generation of induced Tfr cells via a PD-L1-dependent mechanism112, suggesting that the role for PD-1 signalling for Tfr cell formation is context-dependent and more complex than originally thought. The main barrier for determining whether inhibiting Tfr cells is a logical strategy to improve vaccination is our lack of knowledge of their precise role in the GC, particularly whether they suppress humoral autoimmunity arising from the GC53. Currently, there are conflicting reports about whether Tfr cells constrain vaccine-specific responses or non-vaccine-specific responses in the GC50–52. For these cells to be a viable target, definitive evidence would be needed to demonstrate that the role of Tfr cells in the GC is to restrain responses to foreign, rather than self, antigens.\n\n\nPotential dangers of enhancing Tfh responses\n\nAlthough it is clear that Tfh cells are essential for a productive response to vaccination, they have also been implicated in a number of autoimmune conditions as key drivers of disease. Sanroque mice have a point mutation in the Roquin1 gene (Rc3h1) that causes a lupus-like phenotype that is driven by Tfh cells that support GCs in the absence of exogenous antigen116,117. As B cells can acquire self-reactivity during somatic hyper-mutation, findings from the sanroque mice suggest that increases in Tfh cell number may lead to a break in GC tolerance, enabling self-reactive B cells to be selected in the GC. Correspondingly, several autoimmune conditions have been associated with an increase in cTfh frequencies54,118–120 or have a skewed cTfh population away from cTfh1 and toward cTfh2 or cTfh17 or both57,118,121,122. This is an important consideration in the context of enhancing Tfh responses to vaccination, particularly in older persons, as the occurrence of autoantibodies increases with age123, suggesting that the B cell pool may contain a higher frequency of autoreactive B cells able to enter the GC. Taken together, these studies demonstrate a potential hazard of increasing Tfh cell frequencies or function in vaccination and indicate that autoantibody production would need to be accounted for in future study design as a possible outcome.\n\n\nConcluding remarks\n\nThe GC is critical for the production of long-lived antibody-secreting plasma cells after vaccination, making it a promising cellular response to improve vaccine efficacy. There are many players in the GC response; Tfh and Tfr cells tightly control its size and output and thus make them key targets to manipulate in vaccine design. Altering vaccines in a way that increases Tfh cell formation or function (or both) or reduces the suppression exerted on the GC by Tfr cells may be a rational strategy to improve vaccine responses. As vaccines need to have an extremely high safety profile, any perturbations to vaccines must be very low-risk. Because of this, the most logical way to manipulate follicular T cells is to use antigen doses or adjuvants that favour differentiation of Tfh cells with excellent B cell helper capacity, and suppress Tfr cell development. Recent research into next-generation adjuvants demonstrates that adjuvants that support enhanced antibody production also associate with increased numbers of Tfh cell in experimental animals. To determine whether this can translate into enhanced Tfh responses in humans, cTfh cells will be a useful biomarker of GC Tfh responses in preliminary clinical trials. Further research in both humans and animal models into precisely how to manipulate Tfh and Tfr cells to improve vaccine responses may enable us to address some of our current unmet clinical requirements for improved vaccines.", "appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests.\n\n\nGrant information\n\nMichelle A. Linterman and Danika L. Hill are supported by funding from the Biotechnology and Biological Sciences Research Council and the European Research Council (637801 TWILIGHT).\n\n\nReferences\n\nAndre FE, Booy R, Bock HL, et al.: Vaccination greatly reduces disease, disability, death and inequity worldwide. Bull World Health Organ. 2008; 86(2): 140–6. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "12009", "date": "20 Jan 2016", "name": "Di Yu", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12010", "date": "20 Jan 2016", "name": "David Tarlinton", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions", "responses": [] }, { "id": "12011", "date": "20 Jan 2016", "name": "Hui 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", "responses": [] } ]	1	https://f1000research.com/articles/5-88
https://f1000research.com/articles/5-86/v1	20 Jan 16	{ "type": "Research Article", "title": "Strong morphological defects in conditional Arabidopsis abp1 knock-down mutants generated in absence of functional ABP1 protein", "authors": [ "Jaroslav Michalko", "Matouš Glanc", "Catherine Perrot-Rechenmann", "Jiří Friml", "Jaroslav Michalko", "Matouš Glanc", "Catherine Perrot-Rechenmann" ], "abstract": "The Auxin Binding Protein 1 (ABP1) is one of the most studied proteins in plants. Since decades ago, it has been the prime receptor candidate for the plant hormone auxin with a plethora of described functions in auxin signaling and development. The developmental importance of ABP1 has recently been questioned by identification of Arabidopsis thaliana abp1 knock-out alleles that show no obvious phenotypes under normal growth conditions. In this study, we examined the contradiction between the normal growth and development of the abp1 knock-outs and the strong morphological defects observed in three different ethanol-inducible abp1 knock-down mutants (abp1-AS, SS12K, SS12S). By analyzing segregating populations of abp1 knock-out vs. abp1 knock-down crosses we show that the strong morphological defects that were believed to be the result of conditional down-regulation of ABP1 can be reproduced also in the absence of the functional ABP1 protein. This data suggests that the phenotypes in abp1 knock-down lines are due to the off-target effects and asks for further reflections on the biological function of ABP1 or alternative explanations for the missing phenotypic defects in the abp1 loss-of-function alleles.", "keywords": [ "Arabidopsis", "auxin", "AUXIN BINDING PROTEIN 1 (ABP1)", "knock-down mutant", "off-target" ], "content": "Introduction\n\nThe naturally occurring auxin, indole-3-acetic acid, plays a central role in plant growth and development alone or in orchestration with other plant hormones. Proper sensing and interpretation of fluctuating cellular auxin signals is necessary for mediating a diverse range of developmental and cell biology responses (Enders & Strader, 2015; Grunewald & Friml, 2010; Paciorek et al., 2005; Petrasek et al., 2006). In the early screens for auxin receptors, Auxin Binding Protein 1 (ABP1) has been identified based on its ability to bind auxin with high affinity (Hertel et al., 1972; Löbler & Klämbt, 1985) and soon became a prime candidate for an extracellular auxin receptor based mainly on electrophysiological studies utilizing antibodies against ABP1 that showed rapid ABP1-mediated modulation of plasma membrane ion transport in an early step of auxin action (Barbier-Brygoo et al., 1989; Leblanc et al., 1999). Over the next decades, the auxin-binding activity of ABP1 has been characterized in detail by biochemical studies (Batt et al., 1976; Napier et al., 2002; Napier & Venis, 1995; Ray et al., 1977) and its protein structure including the auxin-binding pocket has been revealed (Woo et al., 2002). Phylogenetic studies have shown that ABP1 homologues are present in the genomes of all plant species from bryophytes to flowering plants (Tromas et al., 2010) with more than one copy present e.g. in the genome of maize, rice, poplar or the moss Physcomitrella patens (http://phytozome.jgi.doe.gov/pz/portal.html).\n\nSince its discovery, however, the biological importance of the ABP1 protein as a plasma membrane auxin receptor has been a matter of debates, in part because of its predominant subcellular localization in the endoplasmic reticulum (ER) in maize where the conditions for auxin binding are unfavorable (Habets & Offringa, 2015; Napier et al., 2002). Recently, these discussions were revived by the isolation of two new Arabidopsis abp1 knock-out alleles, abp1-c1 and abp1-TD1 (Gao et al., 2015) that show no obvious phenotypes under standard growth conditions. The contradiction between this observation and the previously published embryo-lethal phenotypes of abp1 mutants (Chen et al., 2001; Tzafrir et al., 2004) has recently been clarified by proving that the embryo-lethality of the originally reported alleles abp1-1 and abp1-1s was caused by disruption of the tightly-linked neighboring gene BELAYA SMERT (BSM) rather than knock-out of ABP1 (Dai et al., 2015; Michalko et al., 2015). This correction and the demonstration of normal embryo development in the abp1 knock-outs (Michalko et al., 2015) suggest that ABP1 plays no essential role in early Arabidopsis embryogenesis.\n\nThe ongoing discussion focuses on the relevance of ABP1 in auxin signaling and other post-embryonic auxin-related biological processes that have been demonstrated using different genetic tools, namely the conditional knock-down (KD) lines, the abp1-5 weak allele harboring a point mutation in the ABP1 auxin-binding pocket and gain-of-function alleles, all of which often provided internally consistent results (Braun et al., 2008; Čovanová et al., 2013; David et al., 2007; Grones et al., 2015; Robert et al., 2010; Sassi et al., 2014; Tromas et al., 2013; Xu et al., 2010; Xu et al., 2014).\n\nConditional ABP1 KD lines SS12S6, SS12K9 and abp1-AS have been generated using two fundamentally different approaches of gene or protein down-regulation. In the SS12S6 and SS12K9 lines, ABP1 was inactivated by inducible over-expression of a recombinant immunoglobulin fragment termed single-chain fragment variable (scFv) (Conrad & Fiedler, 1998). This construct, consisting of the heavy- and light-chain variable domains of a well-characterized anti-ABP1 monoclonal antibody mAb12 (David & Perrot-Rechenmann, 2001; David et al., 2007; Leblanc et al., 1999) linked by a flexible peptide was additionally fused to the sequence encoding the 3‘KDEL motif to mediate scFv ER-retention in the SS12K9 line, while the SS12S-encoded scFv12 was meant to be secreted to the apoplast. In planta-produced scFv12 was able to pull down ABP1, and reciprocally immuno-precipitation of ABP1 using another antibody was shown to pull down scFv12 (Tromas et al., 2009). An antisense approach was utilized in the abp1-AS line, where inducible over-expression of full-length ABP1 antisense cDNA led to the formation of duplexes with its sense mRNA, thus preventing ABP1 translation, and potentially also transcription by RNA interference mechanism (Meister & Tuschl, 2004; Tufarelli et al., 2003). Both antibody- and antisense-based lines use the ethanol-inducible system, which is well established and widely used for the conditional expression of plant genes (Deveaux et al., 2003; Roslan et al., 2001).\n\nThese three abp1 knock-down lines have been instrumental to connect ABP1 function to multiple cellular and developmental processes. For example, they showed defects in shoot and root growth (Braun et al., 2008; Tromas et al., 2009), cell wall re-modeling (Paque et al., 2014) or clathrin-mediated endocytosis of PIN auxin efflux carriers (Dhonukshe et al., 2007; Robert et al., 2010). In contrast, the abp1 gain-of-function transformants promote PIN internalization both in tobacco and Arabidopsis (Grones et al., 2015; Robert et al., 2010). Contrasting effects of ABP1 KD and gain-of-function lines were shown also in the case of auxin effect on the control of leaf epidermal pavement cells morphogenesis (Braun et al., 2008; Nagawa et al., 2012) on ROP GTPase activation (Xu et al., 2010) and on microtubule rearrangement (Chen et al., 2014; Xu et al., 2014). Furthermore, analysis of ABP1 variants with mutations in the auxin-binding pocket demonstrated the importance of auxin-binding to ABP1 for its gain-of-function phenotypes (Grones et al., 2015). Altogether, these studies provided an internally consistent picture showing involvement of ABP1 signaling in multiple physiological and cellular processes. These observations were further supported by the finding that loss-of-function mutants in TMK receptor-like protein kinases, that were recently shown to interact with ABP1 in an auxin-inducible manner, show similar phenotypes with abp1 KD mutants (Xu et al., 2014) which was consistent with the importance of the ABP1/TMK complex-mediated auxin perception in plant development. Recent identification of wild-type looking Arabidopsis abp1 loss-of-function alleles by Gao et al. (2015) thus questions the interpretation of data obtained in the aforementioned studies.\n\nHere, we address the missing phenotypes in the true abp1 null alleles in relation to the strong and consistent morphological defects observed in the conditional abp1 knock-down lines. We show that the morphological phenotypes in SS12S6, SS12K9 and abp1-AS can be generated in the absence of functional ABP1 protein and we discuss possible underlying causes of this.\n\n\nMaterial and methods\n\nArabidopsis thaliana mutants used in this study were: abp1-c1, abp1-TD1 (Gao et al., 2015), abp1-AS, SS12K9, SS12S6 (Braun et al., 2008; David et al., 2007). A. thaliana Col-0 wild type seeds were obtained from The Nottingham Arabidopsis Stock Centre (NASC, http://www.arabidopsis.info). For in vitro experiments, seeds were surface-sterilized with chlorine vapor, vernalized for 2 days in the dark at 4°C and grown on 1/2 MS 0.8% agar medium with or without 1% w/v sucrose (pH 5.9) on vertical Petri dishes under long day conditions (16 h light/8 h dark) or in complete darkness at 21°C. A sterilized microtube with 500 µl 5% ethanol was placed at the bottom of the plate to induce expression of abp1-AS, SS12K9 and SS12S6 constructs before germination. Plates with 5-day old etiolated or 7-day old light-grown seedlings were scanned on a flatbed scanner, phenotyped by visual examination and used for DNA/RNA extraction.\n\nEthanol-inducible ABP1 down-regulating lines (abp1-AS, SS12K9, SS12S6) were genotyped for the presence of the alcR gene encoding the transcriptional regulator of the ethanol-inducible system using primers alcR_for and alcR_rev (Table 1). Fragments amplified from abp1-c1 with primer pairs ABP1-U409F + ABP1-586R or ABP1-5P + ABP1-586R were digested with BslI, which cuts the WT fragment once and does not cut the mutant fragment; abp1-TD1 was genotyped as described previously (Gao et al., 2015). Genomic DNA was isolated using the CTAB extraction method. GoTaq G2 polymerase (Promega) and Bio-Rad T100 Thermal Cycler were used for PCR under following conditions: initial denaturation 5 min 98°C; 35–45 cycles (denaturation 30 s at 98°C; annealing 30 s at 55°C, elongation 1 min at 72°C); final elongation 5 min at 72°C. Restriction analysis was performed by adding the restriction enzyme directly to unpurified PCR reaction. Alternatively, Phire Plant Direct PCR Kit (Thermo Scientific by Finnzymes) and QIAquick Gel Extraction Kit (QIAGEN) were used following manufacturer’s instructions to genotype the SS12K9 x abp1-c1 line.\n\nTotal RNA from approximately twenty 8-day old seedlings frozen in liquid nitrogen was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and purified using RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions. 2 µg of purified total RNA were used for a reverse transcription reaction using the iScript cDNA Synthesis Kit (BioRad). qRT-PCR was performed using the LightCycler 480 SYBR Green I Master chemistry (Roche) in a LightCycler480 II thermal cycler (Ser. no. 5659, Roche) according to manufacturer’s instructions. cDNA diluted 1:10 in water was used as a template to prepare 5 µL reaction mixture (final volume). Primers used for the quantitative RT-PCR were designed using QuantPrime (http://www.quantprime.de). The ABP1 cDNA fragment (84 bp in length) was amplified with ABP1-2E and ABP1-586R primers. Arabidopsis Tubulin beta chain 2 (TUB2, At5g62690) amplified with TUB2-F and TUB2-R primers was used as a reference gene (Dataset 1). Gene expression was calculated with the 2-ΔΔCT method (Livak & Schmittgen, 2001). Results are expressed as the average +/- standard deviation of 2 biological and three technical replicates. Sequences of primers used for genotyping and qRT-PCR analysis are listed in Table 1.\n\n\nResults\n\n(A) abp1-AS × abp1-c1 F2 plants grown for 7 days in the presence of 5% ethanol segregate strong morphological defects characteristic of the abp1 conditional knock-down (KD) alleles approximately in a 3:1 ratio. (B) alcR-specific PCR bands amplified from the genomic DNA of abp1-AS × abp1-c1 F2 plants shown in (A) demonstrate that the KD phenotype is caused by the presence of the ethanol-inducible insertion. (C) Phenotypes of the scFv12-based KD lines segregate similarly in F2 crosses with abp1-c1, while altered segregation ratios can be observed in F2 of all three KD alleles crossed to abp1-TD1, which is most apparent in seedlings grown for 5 days in the dark (grey background).\n\n(A) Representative abp1-AS × abp1-c1 F2 plants, (B) PCR products amplified from their genomic DNA and (C) segregation ratios from all crosses show that the ethanol-inducible phenotypes segregate independently of the presence of abp1 knock-out alleles following approximately Mendelian rules for di-hybrid crosses. Homozygous abp1 knock-out mutants with the inducible KD phenotype could be found in all crosses (plants 2,5,8 in (A) and (B), red numbers in (C)), suggesting that the phenotype does not require a functional ABP1 gene. Strong deviations from the expected Mendelian segregation were detected in the SS12K9 × abp1-c1 cross, indicating genetic linkage between ABP1 locus and the inserted ethanol-inducible scFv construct.\n\n(A) Representative seedlings of the ethanol-induced F3 progeny of one of the SS12K9 × abp1-c1 F2 plants (plant A) that showed KD phenotype in the absence of the functional ABP1. All F3 seedlings manifesting KD phenotype were homozygous for abp1-c1 mutation. (B) Genotyping of the plants shown in A. The image is assemled from different regions of two gels that were copy-pasted next to each other in order to save space. (C) qRT-PCR analysis of KD-phenotype positive F3 seedlings of both lines revealed that ABP1 transcript levels are reduced by about 80% like in the original abp1-c1 mutant. Altogether these data confirm that in the abp1 down-regulating lines the KD phenotype can be manifested without the ABP1 function. In (C) average of two biological and three technical replicates +/- SD is shown. PC- positive control.\n\nTo investigate the contradiction between missing phenotypic defects in the loss-of-function abp1 alleles and strong morphological defects of conditional ABP1 down-regulating lines (knock-down; KD), we decided to cross both types of lines to test three possible scenarios: 1) The absence of the strong morphological defects in the abp1-c1 or abp1-TD1 alleles is caused by an adaptation of the plants to the permanent loss of the ABP1 function, which compensates for this deletion; 2) the strong morphological phenotypes induced in the KD lines do not require functional ABP1 and are caused by off-target effects; or 3) both abp1-TD-1 and abp1-c1 lines contain background mutation(s) that suppress the phenotypes caused by the absence of ABP1.\n\nWe crossed each of the conditional lines with abp1-TD1 and abp1-c1 null mutants and with an ABP1-WTc1 line as a control and analyzed seedling phenotypes of ethanol induced F2 segregating plants (Figure 1a). We hypothesized that in case of an adaptive process, the conditional abp1 KD phenotypes (short wavy roots and epinastic cotyledons) would not be manifested in homozygous abp1 null background, resulting in a 9/16 KD and 7/16 WT phenotype segregation ratio. If the inducible phenotypes in the KD lines are independent of ABP1, these phenotypes will be manifested even in the absence of the functional ABP1 gene, thus resulting in a classic Mendelian 3/4 KD and 1/4 WT phenotype segregation ratio. In case of the presence of background suppressive mutation(s), the KD phenotype segregation ratio would lie anywhere between 3/16 (dominant suppressor mutation closely linked to the ABP1 locus) and 3/4 (recessive mutation with low penetrance and no linkage to ABP1) (Supplementary Figure 1).\n\nSegregation of the morphological phenotypes in the F2 plants from different crosses is summarized in Figure 1b. These observations show that strong phenotypes in both the abp1 antisense-based and the scFv12-based conditional knock-down lines segregate approximately 75% in the F2 crosses with abp1-c1. This observation favors the scenario that the strong morphological defects in the KD lines are not influenced by the presence or absence of the functional ABP1 gene copy. The F2 phenotypic segregation is however shifted in favor of WT-looking plants in all three KD lines crossed to abp1-TD1. This segregation shift may be ascribed to partial transcriptional silencing of the ethanol-inducible constructs due to the presence of multiple 35S promoters/enhancers in the constructs themselves as well as the tandem T-DNA insertion in abp1-TD1.\n\nWe genotyped all analyzed F2 plants for the presence of the alcR transcriptional regulator, which is an integral part of the ethanol-inducible system and verified that the observed morphological defects were indeed correlating with the presence of the ABP1 KD constructs (Figure 1c). About 5% of seedlings from all lines showed WT phenotype despite being positive for the presence of alcR or vice versa. As this phenomenon was independent of ABP1 genetic background and could not be reproduced in F3 progeny (Supplementary Figure 2), we put it down to biological variability and/or occasional silencing of the ethanol-inducible constructs.\n\nTo investigate whether the abp1 KD phenotypes can be observed in the absence of a functional copy of the At4g02980 ABP1 gene we further genotyped the respective abp1 mutations in F2 seedlings of all crosses (Figure 2). As summarized in Figure 2c, in all crosses we were able to identify multiple homozygous abp1 mutants that showed the strong KD phenotype following ethanol induction. This analysis demonstrates that strong morphological phenotypes in abp1 antisense-based (abp1-AS) and scFv12 antibody-based (SS12S6, SS12K9) conditional KD lines can be generated also in a null abp1 background.\n\nIn case of the crosses SS12K9 × abp1-c1 and SS12K9 × abp1-TD1 we observed a lower level of allelic segregation between the abp1 mutations and the KD construct in their F2 progeny (Figure 2c). Out of 28 genotyped plants with WT phenotype, 24 (85.7%) were homozygous for abp1 mutation and did not contain the ethanol-inducible KD cassette. These results point towards genetic linkage between these two loci, most likely caused by the positional effect of the KD cassette located close to the ABP1 locus on the chromosome 4. Nevertheless, some level of genetic recombination was happening between the two loci in the crosses as demonstrated by the identification of three F2 SS12K9 × abp1-c1 plants showing KD phenotype that were homozygous for abp1-c1 mutation (Figure 2c). This analysis confirms that also SS12K9 conditional KD construct can generate strong morphological phenotypes in the homozygous abp1 knock-out alleles despite the insertion position being linked to the ABP1 locus. Altogether these data are consistent with results obtained by the other crosses and further support that morphological phenotypes in the abp1 knock-down lines can be generated in the absence of the functional ABP1.\n\nNext we tested the occurrence of the strong KD-induced morphological phenotypes in the absence of the functional ABP1 in the next generation by analyzing the F3 progeny of two SS12K9 × abp1-c1 plants showing strong KD phenotype. We confirmed that the F3 progeny was homozygous for the abp1-c1 mutation and segregated the ethanol-inducible construct approximately in a 3:1 ratio (Figure 3b). After induction with ethanol, the analyzed F3 population of the SS12K9 × abp1-c1 plant A segregated into 27 plants (67.5%) with KD phenotype and 13 WT looking plants (32.5%) (Figure 3). The F3 population of plant B segregated into 18 plants with KD phenotype (81.2%) and 4 WT looking plants (18.2%) (data not shown). Genotyping of all F3 plants with ethanol-inducible phenotypes revealed that they contain KD construct in the homozygous abp1-c1 background (Figure 3b). Notably, among the 17 analyzed WT looking F3 seedlings we also identified two plants that contain the ethanol-inducible construct in homozygous abp1-c1 background (Figure 3b) suggesting that in these plants the functionality of the construct was affected, most probably by its silencing. Nonetheless, the majority of the plants containing the ethanol-inducible construct generated the strong morphological phenotypes even in the abp1-/- homozygous background.\n\nWe also analyzed the ABP1 expression in WT, abp1-c1 and SS12K9 × abp1-c1 F3 seedlings by quantitative RT-PCR just to verify that introducing KD alleles does not influence, in any way, the ABP1 expression (Figure 3c). We observed ca. 80% decrease in ABP1 transcript levels in abp1-c1. We assume that this difference - somewhat surprising, since the CRISPR-induced small deletion does not necessarily decrease transcript levels - is probably caused by the decreased stability of the mutant mRNA. SS12K9 × abp1-c1 F3 plants positive for the KD phenotype and homozygous for abp1-c1 showed the same 80% decrease in ABP1 transcription.\n\nIn summary, the phenotypic, genotypic and expression analyses consistently showed that all three conditional abp1 knock-down alleles can generate strong morphological defects also in the absence of the functional ABP1 protein.\n\n\nDiscussion\n\nAll three available conditional abp1 knock-down alleles have been extensively characterized and used to link number of developmental and cellular processes to the ABP1-mediated signaling (for overview, see Grones & Friml, 2015). They are based on two unrelated strategies for down-regulation of the protein’s functionality: the antisense (abp1-AS) and the scFv12 monoclonal antibody expression (SS12S6, SS12K9), which suppress the protein functionality by entirely different mechanisms and at different levels (Tromas et al., 2009). All three lines showed consistent and reproducible results in a number of different laboratories and a number of developmental, physiological and cellular processes.\n\nNonetheless, our analysis, made possible by the newly available abp1 knock-out lines (Gao et al., 2015), strongly suggests that these observed and described effects are not caused by conditional down-regulation of the ABP1. This is supported by the fact that all three constructs show the same strong conditional phenotypes in two different homozygous abp1 null alleles. This means that even in the absence of the functional ABP1 protein, the ethanol-inducible constructs are inducing phenotypic defects that were originally ascribed to the down-regulation of ABP1. Therefore, results generated using these lines need to be critically re-interpreted.\n\nAll three types of abp1 KD Arabidopsis lines generate indistinguishable morphological phenotypes. How it is possible that independent lines using fundamentally different approaches for functional down-regulation of a unique target would have in fact the same off-target effects; we do not know. One possible explanation is that the morphological defects are an artifact of the ethanol-inducible expression system. However, control lines generated in parallel using the same vector and expressing the UIDA reporter gene did not exhibit any significant growth and developmental alterations (Braun et al., 2008). Furthermore, a number of authors have used the same ethanol-inducible system to control the expression of distinct genes and to the best of our knowledge, there are no reports describing similar phenotypes by using the ethanol-inducible system for other genes in other studies (Battaglia et al., 2006; Deveaux et al., 2003; Laufs et al., 2003; Peaucelle et al., 2008; Roslan et al., 2001). This system was also used to successfully rescue mutant defects after ethanol induction of gene expression e.g. for LEAFY (Maizel & Weigel, 2004) or for N-myristoyltransferase (Pierre et al., 2007) indicating that it is not responsible per se of the phenotypes observed with the ethanol inducible ABP1 AS and scFv12 constructs. In tobacco plants and BY-2 cells, tetracycline de-repressible promoter-driven expression of the SS12S and SS12K constructs resulted in similar growth defects as their ethanol-inducible expression in Arabidopsis (Braun et al. 2008; David et al., 2007), suggesting that the observed phenotypes are tightly correlated to the scFv12 action. The expression of the scFv12 in the cytosol had however no effect on cell proliferation in BY2 cells indicating that expression of scFv12 per se is not sufficient to generate severe phenotypes whatever its cellular localisation and that scFv12 effects are correlated to its secretion and/or retention in the ER that are known location of ABP1 (David et al., 2007).\n\nAnother possibility is that both the antisense- and antibody-based lines have off-target(s) either on the very same gene(s) or elements of a common genetic pathway. Such a hypothesis would be supported by strict similarities in the phenotypes resulting from ABP1 antisense and scFv12 expression and by the fact that opposite and auxin-related defects were observed in both constitutive and conditional gain-of-function Arabidopsis transgenic plants as well as transitionally expressing tobacco cells (Grones et al., 2015; Robert et al., 2010). ABP1 is placed within the superfamily of cupins based on the presence of cupin-like motifs HXH(X)11G and P(X)4H(X)3N (where X is any amino-acid residue) and a β-barrel jellyroll fold subunit structure (Dunwell et al., 2004; Woo et al., 2002). The epitope recognized by the scFv12 might be present in proteins belonging to this functionally highly diverse protein superfamily. On the other hand, the sequence similarity of even the closest ABP1 homologues in Arabidopsis does not seem to be sufficiently high to be targeted by the abp1-AS constructs, thus this explanation is unlikely as well.\n\nWe also cannot completely rule out that the WT phenotype of the abp1 knock-out mutants is caused by suppressor mutation(s). However, we do not consider it very likely, as this would imply that the similar mutation(s) or mutations with similar effects are present in the genetic background of both abp1-c1 and abp1-TD1, which are independent alleles from independent mutant collections.\n\nIn summary, we do not understand how it is possible that the used abp1 knock-down alleles generate the similar strong morphological phenotypes also in absence of the functional ABP1 protein. All possible explanations we could come up with are unlikely, including common off-targets in abp1 antisense and antibody KD lines or common suppressor mutations in two different abp1 knock-out alleles. Thus, more experimentation is needed to figure out what really happens in the different abp1 KD lines and how it is possible that they independently generate phenotypes that are so consistent. Whatever the explanation at the end will be, in light of the presented data it seems obvious that these lines do not act solely by down-regulating the ABP1 function, despite the accumulation of well-fitting data from independent and complementary approaches. It is a sobering realization that even when you use independent approaches with all standard controls performed, there is no real guarantee that the observations will not lead you amiss.\n\n\nData availability\n\nF1000Research: Dataset 1. Scans of ethanol-induced F2 seedlings of crosses (A) SS12S6 × abp1-c1, (B) SS12S6 × abp1-TD1, (C) abp1-AS × abp1-c1, (D) abp1-AS × abp1-TD1, (E) SS12K9 × abp1-c1 and (F) SS12K9 × abp1-TD1 that were used for phenotyping and genotyping (Figure 1 and Figure 2)., 10.5256/f1000research.7654.d110722 (Michalko et al., 2016a).\n\nF1000Research: Dataset 2. Agarose gel images from the PCR genotyping of the F2 crosses (A) SS12S6 × abp1-c1, (B) SS12S6 × abp1-TD1, (C) abp1-AS × abp1-c1, (D) abp1-AS × abp1-TD1, (E) SS12K9 × abp1-c1 and (F) SS12K9 × abp1-TD1 (Figure 3), 10.5256/f1000research.7654.d110723 (Michalko et al., 2016b).\n\nF1000Research: Dataset 3. Source qPCR data (Figure 3c), 10.5256/f1000research.7654.d110724 (Michalko et al., 2016c).", "appendix": "Author contributions\n\n\n\nJF, JM, CP and MG designed the experiments and wrote the manuscript, JM and MG performed most experiments and analyzed the data. All authors have seen and agreed to the final content of the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by ERC Independent Research grant (ERC-2011-StG-20101109-PSDP to JF). JM internship was supported by the grant “Action Austria – Slovakia”. MG was supported by the scholarship \"Stipendien der Stipendienstiftung der Republik Österreich\". Work by EH and CPR were supported by ANR blanc ANR-14-CE11-0018.\n\n\nAcknowledgements\n\nWe would like to thank Mark Estelle and Yunde Zhao for providing abp1-c1, abp1-TD1 and abp1-WTc1 seeds. We thank Emeline Huault for technical assistance.\n\n\nSupplementary material\n\nExpected genotype and phenotype segregation ratios for three possible scenarios are shown. S/s = ethanol-inducible cassette positive/negative, A/a = wild-type ABP1/abp1 knock-out. Genotypes manifesting wild-type phenotype are shown on white background, KD phenotype on green background, genotypes that might exhibit both WT and KD phenotypes are on pale green background.\n\nIn the presence of 5% ethanol, F3 progeny show homogenous KD phenotype indicating that silencing of the construct might be responsible for wild-type phenotype of this plant in the F2 generation.\n\n\nReferences\n\nBarbier-Brygoo H, Ephritikhine G, Klämbt D, et al.: Functional evidence for an auxin receptor at the plasmalemma of tobacco mesophyll protoplasts. Proc Natl Acad Sci U S A. 1989; 86(3): 891–895. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBatt S, Wilkins MB, Venis MA: Auxin binding to corn coleoptile membranes: Kinetics and specificity. Planta. 1976; 130(1): 7–13. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "12006", "date": "26 Jan 2016", "name": "Lars Ostergaard", "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, Michalko et al. investigates the basis of the developmental phenotypes observed in knock-down (KD) lines of the Arabidopsis ABP1 gene. By analysing segregation of defects in root elongation in the F2 and F3 generation of crosses between three KD abp1 alleles and two recently reported abp1 knock-out (KO) alleles, they conclude that the previously reported phenotypes of the KD lines are not due to loss of ABP1 function. The manuscript is clearly written and the experiments are thoroughly carried out providing an important contribution to the ABP1 saga.Although I sympathise with the authors difficulty in identifying an explanation for how the KD lines can lead to the observed abnormalities, one experiment that should be done would in my opinion be a test of the expression of the closest ABP1-like genes that can be identified.", "responses": [] }, { "id": "12004", "date": "05 Feb 2016", "name": "Richard M. Napier", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 produced a very thorough analysis of a set of well-known conditional ABP1 knock-down Arabidopsis lines. Using crosses with the recently-described abp1 knock-out lines from the work of Gao et al. (2015), the manuscript reveals that the phenotypes previously associated with loss of ABP1 can be induced even in genetic backgrounds that lack ABP1. These findings cover results from the two independent strategies used to create the conditional knock-down phenotypes, conditional immunosuppression and conditional antisense expression.The assay used for this work was Arabidopsis root growth, not the very detailed phenotypes described recently from the conditional lines such as PIN endocytosis, ROP GTPase activation or microtubule rearrangement. However, impaired root growth was part of the initial suite of phenotypes associated with induced knock-down. The assays show very clearly that growth impairment correlates with the inheritance of the inducible cassette even in the absence of a functional ABP1 gene. We can surmise that the detailed subcellular phenotypes listed above are associated with the gross morphological changes recorded here. There is clearly some intriguing physiology associated with the switch induced by ethanol treatment, but ABP1 is not part of that story. The title and abstract are appropriate (but see note below), the work is done well, the data is presented clearly and fully, the text is very well structured and is easy to follow. There is a thorough introduction which explains how and why experimentation developed to tackle tangible problems linked to existing knowledge and understanding of ABP1 genetic resources. It is shown that the science was not misled by carelessness or device, that a spectrum of consistencies coloured the hypotheses covering ABP1 activity, and that these hypotheses have now been proven incorrect thanks to improved technologies, vigilance and critical reappraisal.The abstract ends with a sentence “…asks for further reflections on the biological function of ABP1 or alternative explanations for the missing phenotypic defects in the abp1 loss-of-function alleles.” I think that this would be better if it read “… AND alternative explanations for the intriguing phenotypes previously associated with loss of ABP1 activity”. Or similar. I understand that there is a requirement on all sides of the debate to be objective and critical about their data and no harm is done by registering this. However, the balance of probabilities needs to be recognised and, in my view, much more has to be gained from encouraging discovery of the root cause of the many fascinating phenotypes thrown up by the ABP1 KD lines than by searching absent defects. Therefore a small rewording at the end of the abstract is recommended. Otherwise I have no changes to suggest.", "responses": [] }, { "id": "12003", "date": "09 Feb 2016", "name": "Christian Luschnig", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn their m/s, the authors present an analysis of genetic interactions between published ABP1 knockdown lines (based on antisense and immune-suppression approaches) and the Arabidopsis ABP1 locus. After crossing three different knockdown lines into recently described abp1 knockout alleles, followed by segregation analysis of resulting F2 and F3 progeny, the authors came to the conclusion that growth defects of their knockdown lines do not depend on a functional ABP1 locus. These findings represent a valuable contribution to ongoing attempts, trying to clarify ABP1 issues. Nevertheless, off-target loci recognized in the knockdown lines remain mysterious. Are there any ABP1-related loci (cupins?), expression of which could be affected by these knockdown lines? Perhaps this could be tested in the abp1-AS line.Phenotypes of the knockdown lines are reminiscent of mutants with altered auxin responses. Did the authors look into expression of some of the characterized auxin-related loci? This could give us a better idea about the genetic determinants, causing the phenotypes in these knockdown lines.", "responses": [] } ]	1	https://f1000research.com/articles/5-86
https://f1000research.com/articles/4-474/v1	05 Aug 15	{ "type": "Observation Article", "title": "Possible identification of CENP-C in fish and the presence of the CENP-C motif in M18BP1 of vertebrates.", "authors": [ "Leos G. Kral" ], "abstract": "The centromeric protein CENP-C is a base component of the kinetochore. This protein, along with CENP-A has been shown to adaptively evolve in a number of animal and plant species. In order to determine if CENP-C also evolves in fish species, I attempted to retrieve fish CENP-C sequences from GenBank. No Teleostei CENP-C sequences were found either by name or by BLASTP searches with the vertebrate CENP-C motif sequence. A number of putative Teleostei protein sequences were identified in GenBank that have homology to the C-terminal cupin domain of vertebrate CENP-C. These proteins only have partial homology to the CENP-C motif, but evidence is presented that makes it likely that these fish proteins are orthologs of CENP-C. Interestingly, it was also discovered that the CENP-C motif sequence is also mostly present in M18BP1 proteins of fish and some other vertebrates but not in mammals. This finding may have implications for CENP-C and M18BP1 assembly in centromeric regions of different vertebrate taxa.", "keywords": [ "CENP-C", "M18BP1", "centromeric proteins", "teleostei", "kinetochore", "CENP-C motif", "cupin domain protein" ], "content": "Introduction\n\nThe kinetochore is a structure that connects chromosomal centromeric DNA to microtubules during mitosis and meiosis1. The centromere is epigenetically defined by the deposition of nucleosomes that contain the histone H3 variant CENP-A2. Centromeric protein CENP-C is required for both the recruitment of new CENP-A to the centromeric region as well as the initial assembly of the kinetochore. The CENP-C protein is generally considered to be ubiquitous in all eukaryotic taxa since homologs of CENP-C have been identified in yeast3 and Drosophila4 as well as many plants and vertebrates5. While CENP-C evolves so rapidly that very little homology is observed between distantly related taxa, a conserved CENP-C motif has been identified across all lineages studied5. This conserved motif should, therefore, be of utility to identify CENP-C orthologs in other species.\n\nCENP-A has been initially shown to evolve adaptively in Drosophila6, in members of the Bressicaceae family7 and more recently in primates8 and in percid fishes9,10. CENP-C has also been shown to evolve adaptively in a number of animal and plant species5 as well as in primates8. In an effort to determine if CENP-C also evolves adaptively in fish species, searches were conducted in GenBank for Teleostei proteins that had been already identified as CENP-C or for genes that had been annotated as coding for CENP-C. No such fish proteins or genes were found. BLASTP searches of just the Teleostei subset of GenBank were performed with the conserved vertebrate CENP-C motif and these too failed to find identified fish CENP-C proteins or genes. However, these searches did identify fish M18BP1, which, as will be discussed below, contains a sequence homologous to the CENP-C motif. A search of the Chondrichthyes for proteins that had been already identified as CENP-C, or for genes that had been annotated as coding for CENP-C, found one gene annotated as CENP-C in the elephant shark (Callorhinchus milii) genome. Interestingly, while this putative shark CENP-C protein contained a cupin domain at the C-terminal end homologous to the cupin domain found at the C-terminal end of other vertebrate CENP-C proteins, the conserved CENP-C motif was not found in the expected location upstream of the cupin domain. However, a region further upstream does have homology to some of the most conserved amino acids of the CENP-C motif.\n\nBLASTP searches of the Teleostei subset of GenBank with the putative shark CENP-C protein sequence identified a number of genes that were primarily homologous to the C-terminal cupin domain. However, none of these fish sequences were annotated as CENP-C in GenBank. Upon closer analysis, as will be discussed below, these C-terminal cupin domain containing fish proteins do contain sequences that are partly homologous to the conserved CENP-C motif and, therefore, these fish genes could be CENP-C orthologs.\n\n\nMethods\n\nStandard BLASTP searches were performed on the NCBI blast server. The vertebrate CENP-C motif NVRRTKRXRLKPLEYWRGERVBY used in BLASTP searches in this study was obtained from an alignment of 25 species including amphibians, reptiles, birds and mammals (Supplementary File S1). Retrieved sequences were aligned with the MUSCLE alignment feature in Geneious (version 6.1) sequence analysis software.\n\n\nResults and discussion\n\nBLASTP searches with the vertebrate CENP-C motif identified CENP-C proteins from a variety of taxa, including plants, but did not identify any CENP-C in fish lineages. It is possible that CENP-C may be absent in fish, but the ubiquity of this protein in other lineages and the central role of this protein in centromeric function make this unlikely. A C-terminal cupin domain protein encoded by a shark gene annotated in GenBank as CENP-C was used to identify homologs in Teleostei genomes by BLASTP. The retrieved teleost fish homologs were annotated as either calponin homology domain containing protein, neurofilament heavy polypeptide-like protein, or myb-like protein. Within vertebrate CENP-C proteins the RxxRxxxxPLxYWxGERxxY sequence defines identities within the CENP-C motif located within about 100 amino acids upstream of the cupin domain (Figure 1). However, within the shark and teleost fish C-terminal cupin domain-containing protein sequences, only some of these CENP-C motif sequence identities were present (Figure 2) and, therefore, unambiguous identity of these proteins as CENP-C was not obvious.\n\n(A) Diagram of human CENP-C. (B) Amino acids that are identical in the CENPC motif in vertebrates in which CENP-C has been identified. (C) Conserved sequence in the CENP-C central region that is homologous to part of the CENP-C motif. Amino acid locations within human CENP-C protein of conserved sequences are indicated at the beginning and end of each sequence.\n\n(A) Diagram of putative Stegastes partitus CENP-C. (B) Alignment of fish central region sequences that contain the conserved RxxxxPxxWW sequence. (C) Alignment of the fish cupin domain proximal sequences that contain the conserved RxxxxPxxWW sequence. Amino acids matching the conserved sequence identities are highlighted in red. Amino acid locations within each species’ protein are indicated at the beginning and end of each sequence.\n\nIn a recent study that examined the interaction between CENP-C conserved domains and CENP-A containing nucleosomes (or nucleosomes containing histone H3 modified with a CENP-A C-terminal tail), Kato et al.11 identified within the conserved central region of CENP-C a RxSxxPSxWW consensus sequence (Figure 1) that is similar to the core portion of the CENP-C motif. Mutations of the arginine to alanine or the tryptophans to alanine in this sequence prevented the binding of this central region to the nucleosomes. So, functionally, the RxxxxPxxWW portion of the central region sequence is important to centromeric binding of CENP-C. Furthermore, mutations of the arginine, tyrosine and tryptophan in the core CENP-C motif RxxxxPxxYW also reduce the binding affinity the CENP-C to the nucleosomes11. A mutation of arginine to alanine in this core portion of the CENP-C motif was previously shown to prevent the binding of Xenopus CENP-C to centromeres12.\n\nAn alignment of the putative shark and teleost fish CENP-C proteins identified two conserved regions that contained the RxxxxPxxWW sequences (Figure 2). The placement of these sequences corresponds roughly to the locations of the central portion and the CENP-C motif of the vertebrate CENP-C (Figure 1). Therefore, it is likely that the combination of the C-terminal cupin domain and the presence of these centromeric nucleosome binding regions in positions generally corresponding to the locations of the central region and the CENP-C motif identifies these teleost genes as possible CENP-C orthologs. It will be necessary, of course, to verify if this protein is actually found at fish centromeres. It should be noted, however, that the distance between the cupin domain and the “CENP-C motif” position is about twice as long in the putative fish CENP-C in comparison to this distance in CENP-C of other vertebrates. It is interesting that the putative shark “CENP-C motif” location lacks the tryptophans of the RxxxxPxxWW sequence and that Poecilia reticulata has a replacement of the first tryptophan in the conserved central region sequence (Figure 2). However, depending on other factors acting in the assembly of the centromere in various taxa, it may be possible that just one of those conserved RxxxxPxxWW sequences may be necessary for centromeric binding of the putative fish CENP-C. Interestingly, no homology to the RxxxxPxxWW portion of the conserved central region is detectable in CENP-C of reptiles and birds.\n\nInterestingly, BLASTP searches of the Teleostei subset of GenBank retrieved centromeric protein M18BP1 sequences. This protein is recruited to centromeres by CENP-C13,14 and along with centromeric proteins Mis18α and Mis18β functions in the recruitment of CENP-A to centromeres15. The M18BP1 protein contains almost the entire vertebrate CENP-C motif in all vertebrates examined except in mammals (Figure 3). It appears that the CENP-C motif sequence is not exclusive to just CENP-C. Since both CENP-C and M18BP1 associate with centromeres and with each other, it is tempting to speculate that what has generally been regarded as a CENP-C motif sequence facilitates the interaction of both of these proteins with centromeric nucleosomes. Furthermore, since mammalian M18BP1 lacks this CENP-C motif, it is possible that mammalian M18BP1 may be more dependent on association with CENP-C to localize to the centromere than the M18BP1 of other vertebrate taxa.\n\nThe vertebrate CENP-C motif sequence identities (Figure 1A) are highlighted in red.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study was supported by a Faculty Research Grant funded by the University of West Georgia.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nSupplementary material\n\nSupplementary file S1. Alignment of the C-terminal portion of vertebrate CENP-C proteins.\n\nThe vertebrate CENP-C motif containing consensus sequence utilized in BLASTP searches spans amino acids 11 to 33 and is highlighted in red.\n\nClick here to access the data.\n\n\nReferences\n\nCheeseman IM: The kinetochore. Cold Spring Harb Perspect Biol. 2014; 6(7): a015826. PubMed Abstract \| Publisher Full Text\n\nBlack BE, Brock MA, Bédard S, et al.: An epigenetic mark generated by the incorporation of CENP-A into centromeric nucleosomes. Proc Natl Acad Sci U S A. 2007; 104(12): 5008–5013. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMeluh PB, Koshland D: Evidence that the MIF2 gene of Saccharomyces cerevisiae encodes a centromere protein with homology to the mammalian centromere protein CENP-C. Mol Biol Cell. 1995; 6(7): 793–807. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHeeger S, Leismann O, Schittenhelm R, et al.: Genetic interactions of separase regulatory subunits reveal the diverged Drosophila Cenp-C homolog. Genes Dev. 2005; 19(17): 2041–2053. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTalbert PB, Bryson TD, Henikoff S: Adaptive evolution of centromere proteins in plants and animals. J Biol. 2004; 3(4): 18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMalik HS, Henikoff S: Adaptive evolution of Cid, a centromere-specific histone in Drosophila. Genetics. 2001; 157(3): 1293–1298. PubMed Abstract \| Free Full Text\n\nCooper JL, Henikoff S: Adaptive evolution of the histone fold domain in centromeric histones. Mol Biol Evol. 2004; 21(9): 1712–1718. PubMed Abstract \| Publisher Full Text\n\nSchueler MG, Swanson W, Thomas PJ, et al.: Adaptive evolution of foundation kinetochore proteins in primates. Mol Biol Evol. 2010; 27(7): 1585–1597. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFountain DM, Kral LG: Isolation and Characterization of the Etheostoma tallapoosae (Teleostei: Percidae) CENP-A Gene. Genes (Basel). 2011; 2(4): 829–840. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbbey HN, Kral LG: Adaptive Evolution of CENP-A in Percid Fishes. Genes (Basel). 2015; 6(3): 662–671. PubMed Abstract \| Publisher Full Text\n\nKato H, Jiang J, Zhou BR, et al.: A conserved mechanism for centromeric nucleosome recognition by centromere protein CENP-C. Science. 2013; 340(6136): 1110–1113. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMilks K, Moree B, Straight AF: Dissection of CENP-C-directed centromere and Kinetochore assembly. Mol Biol Cell. 2009; 20(19): 4246–4255. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoree B, Meyer CB, Fuller CJ, et al.: CENP-C recruits M18BP1 to centromeres to promote CENP-A chromatin assembly. J Cell Biol. 2011; 194(6): 855–871. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDambacher S, Deng W, Hahn M, et al.: CENP-C facilitates the recruitment of M18BP1 to centromeric chromatin. Nucleus. 2012; 3(1): 101–110. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFujita Y, Hayashi T, Kiyomitsu T, et al.: Priming of centromere for CENP-A recruitment by human hMis18alpha, hMis18beta, and M18BP1. Dev Cell. 2007; 12(1): 17–30. PubMed Abstract \| Publisher Full Text" }	[ { "id": "10498", "date": "25 Sep 2015", "name": "Tatsuo Fukagawa", "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 centromere protein CENP-C is well known as an essential component for functional kinetochore assembly. Due to importance of this molecule, CENP-C must be conserved in Fish species. The author performed BLASTP searches with the conserved CENP-C motif sequence, but any CENP-C homologues in Fish lineages were not identified with this sequence. However, as there is a putative CENP-C sequence in shark genome, BLASTP searches were carried out with C-terminal domain sequence of putative shark CENP-C. Then, the author identified CENP-C candidates from various teleost genomes. Although the author does not show that candidate proteins localize to centromeres, these candidates contains related sequences of CENP-C motif, which were a little divergent from the vertebrate consensus sequences. Interestingly, the authors found that various vertebrate M18BP1s, which are recruited to centromeres by CENP-C, contain the CENP-C motif sequence, but mammalian homologues do not contain the motif sequence. This is an observation article and finding of the CENP-C motif in the M18BP1 sequence is interesting. However, to improve the quality of the paper, the author should revise the manuscript. My specific concerns are following. As the author recognizes, it is necessary to verify whether CENP-C candidates from teleost genome really localize to Fish centromeres. As the author obtained a candidate from Zebrafish genome, such an experiment is not difficult with the Zebrafish experimental system. If the author added localization data, Figure 2 would be interesting. Related to Figure 2. If the author shows sequence comparison of Cupin domain in teleost sequences, it would be helpful. When the author discuss about central region of human CENP-C, it may be better to cite a recent paper by Nagpal et al. (Mol. Biol. Cell, 2015), which says that central region sequence does not exist in chicken CENP-C. Then, the author can emphasize that the central motif is really important for CENP-A binding. The author described some results of the analysis in the Introduction. This is not necessary and it would be better to cut of redundant description.", "responses": [ { "c_id": "1765", "date": "20 Jan 2016", "name": "Leos Kral", "role": "Author Response", "response": "I have revised the manuscript to address concerns #2, #3 and #4. Unfortunately, I do not have the resources to carry out the localization experiment (concern #1). My main motivation in publishing these observations is to bring awareness of this issue to individuals who may have the resources and interest to follow up with the relevant experiments." } ] } ]	1	https://f1000research.com/articles/4-474

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